RESEARCH ARTICLE
Filamin A C- e minal agmen modula es O ai1 exp ession by inhibi ion o
p o ein deg ada ion
Al a o Macias-Díaz,
1
Joel Nie o-Felipe,
1
Isaac Ja dín,
1
Ped o J. Camello,
1
E a M. Ma inez-Quin ana,
4
Gines M. Salido,
1
Ta ik Smani,
2,3
Jose J. Lopez,
1
and Juan A. Rosado
1

1
Depa men o Physiology (Cellula Physiology Resea ch G oup), Ins i u e o Molecula Pa hology Bioma ke s (IMPB),
Uni e si y o Ex emadu a, Cace es, Spain;
2
G oup o Ca dio ascula Pa hophysiology, Ins i u e o Biomedicine o Se ille,
Uni e si y Hospi al o Vi gen del Rocío/Uni e si y o Se ille/CSIC, Se ille, Spain;
3
Depa men o Medical Physiology and
Biophysics, Facul y o Medicine, Uni e si y o Se ille, Se ille, Spain; and
4
Pa hology Se ice, Uni e si y Hospi al o Cace es,
Cace es, Spain
Abs ac
Filamin A (FLNA) is an ac in-binding p o ein ha has been epo ed o in e ac wi h STIM1 modula ing he ac i a ion o O ai1 channels.
Clea ing o FLNA by calpain leads o a C- e minal agmen ha is in ol ed in a a ie y o unc ional and pa hological e en s, including
p o-oncogenic ac i i y in di e en ypes o cance . He e, we show ha ull-leng h FLNA is down egula ed in samples om pa ien s wi h
colon cance as well as in he adenoca cinoma cell line HT-29. This is consis en wi h an inc eased calpain-dependen FLNA clea ing
wi h enhanced exp ession o he C- e minal FLNA agmen accompanied by enhanced exp ession o O ai1 and STIM1, as well as
s o e-ope a ed Ca
2þ
en y (SOCE). To u he explo e he mechanism unde lying he enhancemen o SOCE by he C- e minal FLNA
agmen , we exp essed in HEK-293 cells he C- e minal FLNA egion encompassing epea s 16–24 (FLNA
16–24
agmen ), which
enhancedbo hO ai1andSTIM1aswellasSOCE.T ans ec iono heFLNA
16–24
agmen a enua es O ai1 and STIM1 p o ein deg ada-
ion,and,specifically, ab oga es O ai1alysosomal deg ada ion and e ains his channel in he plasma memb ane. Howe e , he
C- e minal FLNA agmen did no induce a de ec able modifica ion in O ai1bdeg ada ion. Due o he ele ance o SOCE in cell physiol-
ogy, ou esul s p o ide e idence o a no el mechanism o he egula ion o Ca
2þ
influx wi h ele an pa hophysiological implica ions.
NOTE & NOTEWORTHY FLNA clea ing by calpain has been obse ed in a a ie y o umo al, including p os a e and colo ec al can-
ce cells, as well as in non umo al cells, leading o a C- e minal agmen encompassing epea s 16–24. Exp ession o he FLNA
16–24
agmen in HEK-293 cells enhances O ai1 and STIM1 exp ession, as well as SOCE, a mechanism media ed by a enua ion o O ai1a
and STIM1 deg ada ion, p o iding e idence o a no el mechanism o he egula ion o SOCE in no mal and malignan cells.
FLNA; O ai1a; O ai1
b
; s o e-ope a ed Ca
2þ
en y
INTRODUCTION
O ai1 is he po e- o ming subuni o he CRAC (Ca
2þ
elease-ac i a ed Ca
2þ
) channels, a highly selec i e Ca
2þ
channel, ubiqui ously exp essed, ha media e s o e-ope a ed
Ca
2þ
en y (SOCE), a majo pa hway o agonis -induced
Ca
2þ
mobiliza ion (1–3). SOCE plays a ele an ole suppo -
ing a a ie y o cellula unc ions, including gene exp ession,
cell p oli e a ion, di e en ia ion, lac a ion, and pla ele agg e-
ga ion (4–7). SOCE is ac i a ed upon discha ge o he in a-
cellula Ca
2þ
s o es, mainly, bu no exclusi ely (8), he
endoplasmic e iculum (ER), leading o dissocia ion o
Ca
2þ
om he Ca
2þ
senso p o eins, STIM1 and STIM2,
wo single-pass ansmemb ane p o eins wi h highly con-
se ed luminal EF-hand mo i s (9). Ca
2þ
dissocia ion om
he EF-hand mo i leads o STIM p o eins oligome iza ion
and ansloca ion o egions close o he plasma mem-
b ane (PM). This, oge he wi h a well-desc ibed con o ma-
ional change, esul s in he associa ion o STIM p o eins
wi h O ai channels in he PM, o ini ia e Ca
2þ
influx (10–
14). Two O ai1 pa alogs, O ai2 and O ai3, ha e been p o-
posed o modula e Ca
2þ
influx h ough he CRAC channels
(15). Fu he mo e, wo O ai1 a ian s ha e been iden ified
in mammalian cells gene a ed by al e na i e ini ia ion o
ansla ion, gi ing ise o he canonical ull-leng h O ai1 p o-
ein, designa ed as O ai1a, which comp ises 301 amino acids,
and a sho a ian , e med O ai1b, ha o igina es om an al-
e na i e ansla ion ini ia ion a ei he me hionine 64 o 71
(16). Al hough bo h a ian s show simila e ficacy in suppo -
ing he highly Ca
2þ
-selec i e I
CRAC
cu en s (17), impo an
unc ional and biophysical di e ences ha e been epo ed
be ween O ai1aand O ai1b.Fo ins ance,al houghO ai1a
A. Macias-Díaz and J. Nie o-Felipe con ibu ed equally o his wo k. T. Smani and J. A. Rosado con ibu ed equally as co-
senio au ho s.
Co espondence: A. Macias-Diaz ([email p o ec ed]); J. J. Lopez ( [email p o ec ed]); J. A. Rosado ([email p o ec ed]).
Submi ed 8 Oc obe 2024 / Re ised 16 Decembe 2024 / Accep ed 30 Decembe 2024
h p://www.ajpcell.o g 0363-6143/25 Copy igh ©2025 The Au ho s. Licensed unde C ea i e Commons A ibu ion CC-BY 4.0.
Published by he Ame ican Physiological Socie y.
C657
Am J Physiol Cell Physiol 328: C657–C669, 2025.
Fi s published Janua y 7, 2025; doi:10.1152/ajpcell.00745.2024
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suppo s he less Ca
2þ
-selec i e s o e-ope a ed cu en I
SOC
and he a achidona e- egula ed cu en I
ARC
(17), O ai1bdoes
no pa icipa e in I
ARC
,andi s oleinI
SOC
is cell- ype-specific
(18). Fu he mo e, in con as o O ai1b,O ai1ais equi ed
o NF-κB ansc ip ional ac i i y (19). Biophysically,
O ai1ais mo e suscep ible o apid Ca
2þ
-dependen inac-
i a ion han O ai1b(17).
Among he p o eins in ol ed in he modula ion o STIM1-
O ai1 in e ac ion and he ac i a ion o SOCE, filamin A
(FLNA) has been epo ed o in e ac wi h STIM1 hus modu-
la ing he ac i a ion o O ai1 channels (20). FLNA is an 280
kDa ac in-binding p o ein ha suppo s o hogonal b anch-
ing o ac in mic ofilamen s and s abilizes he co ical ac in
ne wo k (21). Human FLNA comp ises an amino- e minal
ac in-binding domain and 24 immunoglobulin (Ig)-like
epea s (22). FLNA is clea ed by calpain in o a 190 kDa ag-
men , con aining he ac in-binding domain and he fi s 15
Ig-like epea s and a second agmen o 110 kDa, comp is-
ing he emaining Ig-like epea s; he la e is u he clea ed
o a 90 kDa agmen (23,24). The sho C- e minal FLNA
agmen has been epo ed o be in ol ed in a a ie y o
unc ional and pa hological e en s. This agmen anslo-
ca e o he nucleus and modula es and ogen ecep o an-
sc ip ional ac i i y (23,24). Fu he mo e, FLNA clea ing
p omo e angiogenesis by acili a ing he nuclea ansloca-
ion o se e al ansc ip ion ac o s (25). In mac ophages,
FLNA C- e minal agmen in e ac s wi h STAT3 and enhan-
ces i s phospho yla ion and nuclea ansloca ion (26). FLNA
C- e minal agmen has been shown o suppo in ahepa ic
cholangioca cinoma p og ession and inhibi ion o calpain
by calpep in impai s cell g ow h in a a ie y o human and
mouse umo s, including human melanoma and p os a e
cance and mouse fib osa coma (27,28). Colo ec al adenoca -
cinoma is among he mos common cance ypes in men and
women. Colo ec al adenoca cinoma cells exhibi enhanced
SOCE associa ed wi h abno mal exp ession o he O ai and
STIM iso o ms and TRPC1 (29), which play a ele an ole in
he de elopmen o di e en cance hallma ks, including cell
mig a ion and su i al and apop osis esis ance (29,30). The
objec i e o his s udy is o in es iga e he unc ional ole o he
FLNA C- e minal agmen in he egula ion o SOCE. He e, we
show ha FLNA is down egula ed in pa ien s wi h colon ade-
noca cinoma and is clea ed bo h in colo ec al adenoca cinoma
HT-29 cells and no mal colon mucosa cells. Inhibi ion o FLNA
clea ing by calpep in in HT-29 cells and exp ession o he
FLNA
16–24
agmen in HEK-293 cells s ongly sugges ha he
FLNA C- e minal agmen is in ol ed in he modula ion o
O ai1 and STIM1 exp ession and he ac i a ion o SOCE.
Enhancemen o O ai1 exp ession by he FLNA
16–24
agmen
in ol es inhibi ion o O ai1adeg ada ion. These obse a ions
p o ide he fi s e idence o he oleo FLNAC- e minal ag-
men in he egula ion o O ai1 exp ession and unc ion.
MATERIALS AND METHODS
Reagen s and An ibodies
Fu a-2 ace oxyme hyl es e ( u a-2/AM) was om Molecula
P obes (Leiden, The Ne he lands). High-glucose Dulbecco’s
modified Eagle’s medium (DMEM), e al bo ine se um, yp-
sin, penicillin/s ep omycin, T izma base, abbi polyclonal
an i-STIM2 an ibody (ca alog numbe STIM2-201AP, epi ope:
amino acids 600–650 o human STIM2), mouse monoclonal
an i-FLNA an ibody [clone: FLMN01 (PM6/317), ca alog num-
be MA5-11705], mouse monoclonal an i-PMCA an ibody
(clone 5F10; ca alog numbe MA3-914, epi ope: amino acids
724–783 o human PMCA; RRID: AB_2061566), Supe Signal
Wes Du a ex ended du a ion subs a e eagen and Pie ce
BCA p o ein assay ki , high-capaci y s ep a idin aga ose
esin, EZ-Link Sul o-NHS-LC-Bio in, and Li e/Dead iabili y/
cy o oxici y ki we e pu chased om The mo Fishe Scien ific
(Wal ham, MA). Comple e EDTA- ee p o ease inhibi o cock-
ail able s (Re e ence name: COEDTAF-RO) we e om Roche
Diagnos ics GmbH (Mannheim, Ge many). Dha maFECT kb
ans ec ion eagen was ob ained om Ho izon Disco e y
(Wa e beach, UK). Thapsiga gin (TG), cycloheximide, calpep-
in [inhibi o o CAPN1 (calpain1)], HEPES [4-(2-hyd oxye hyl)
pipe azine-1-e hanesul onic acid], EGTA [e hylene glycol-bis
(2-aminoe hyle he )-N,N,N0,N0- e aace ic acid], EDTA (e hyl-
enedini ilo e aace ic acid), bo ine se um albumin (BSA), so-
dium azide, dime hyl-BAPTA, sodium asco ba e, bafilomycin
A1, MG132, abbi polyclonal an i-O ai1 an ibody (ca alog
numbe O8264, epi ope: amino acids 288–301 o human
O ai1), abbi polyclonal an i-O ai1 (AB-1) an ibody (ca alog
numbe AV50117, epi ope: amino acids 2–61 o human O ai1),
mouse monoclonal an i-phospho-Filamin-A (Se 2152) an i-
body (Clone PS2,ca alog numbe MABN1834), and abbi poly-
clonal an i-b-ac in an ibody (ca alog numbe A2066, epi ope:
amino acids 365–375 o human b-ac in; RRID: AB_2816311)
we e ob ained om Millipo eSigma (Bu ling on, MA). Rabbi
polyclonal an i-O ai2 an ibody (ca alog numbe TA306419,
epi ope: sequence localized in he C- e minal egion; RRID:
AB_2040046) was om O igene (Rock ille, MD). Mouse mono-
clonal an i-O ai3 an ibody (Clone EPR22575-17; ca alog num-
be ab254260; RRID: AB_2530307) was ob ained om Abcam
(Camb idge, UK). Mouse monoclonal an i-GOK/STIM1 an i-
body(Clone44/GOK;ca alognumbe 610954,epi ope:amino
acids: 25–139 o human STIM1; RRID: AB_398267) was pu -
chased om BD Biosciences (San Jose, CA). Ho se adish pe -
oxidase-conjuga ed goa an i-mouse immunoglobulin G
(IgG) an ibody (RRID: AB_10015289) and goa an i- abbi
IgG an ibody (RRID: AB_2337913) we e om Jackson
Labo a o ies (Wes G o e, PA). N-glycosidase F (PNGase F)
om Elizabe hkingia mi icola was om New England
Biolabs (Ipswich, MA). Fluo escen goa an i- abbi IgG
S a B igh Blue 700 (RRID: AB_2721073; Ca alog numbe
12004161) and goa an i-mouse S a B igh Bue 700 (RRID:
AB_2884948; Ca alog numbe 12004158) an ibodies we e
om Bio-Rad Labo a o ies, Inc. (He cules, CA). pEGFP-N1-
O ai1a-eGFP and pEGFP-N1-O ai1b-eGFP plasmids we e kindly
p o ided by Mohamed T ebak (Depa men o Pha macology
and Chemical Biology, Uni e si y o Pi sbu gh, Pi sbu gh, PA).
pDsRed-Monome -C1-FLNA
16–24
encoding C- e minal FLNA
epea s 16–24 was a gi om Xiaowei Zheng (Depa men o
Molecula Medicine and Su ge y o Ka olinska Ins i u e , Solna,
Sweden). pcDNA3-myc FLNA WT (Addgene No. 8982; h p://
n2 .ne /addgene:8982; RRID: Addgene 8982) and pcDNA3-myc
FLNA S2152A (Addgene No. 8983; h p://n2 .ne /addgene:
89823; RRID: Addgene 8983) plasmids we e a gi om John
Blenis. STIM1-mChe y was a gi om Ch is oph Romanin
(Ins i u e o Biophysics, Johannes Keple Uni e si y Linz).
All o he eagen s we e o an analy ical g ade.
FLNA CLEAVING MODULATES O ai1 EXPRESSION
C658 AJP-Cell Physiol doi:10.1152/ajpcell.00745.2024 www.ajpcell.o g
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Cell Cul u e and T ans ec ion
Non umo al NCM460 (RRID: CVCL_0460) and colo ec al
adenoca cinoma HT-29 cells (RRID: CVCL_0320) we e kindly
p o ided by Ca los Villalobos [Ins i u e o Molecula Biology
and Gene ics (IBGM), Valladolid, Spain]. Colo ec al adeno-
ca cinoma Caco-2 cells (RRID: CVCL_0025) we e p o ided by
Ma io Es e ez (Uni e si y o Ex emadu a, Cáce es, Spain).
Colo ec al ca cinoma HCT116 cells (RRID: CVCL_B7PT) we e
pu chased in ATCC (LGC S anda ds S.L.U.—Spain O fice,
Ba celona, Spain). Cells we e egula ly checked o con amina-
ion. CRISPR-gene a ed O ai1-knockou HEK293 cells (RRID:
CVCL_0045) (HEK-293 O1-KO), STIM1, and STIM2 double-
knockou HEK-293 cells (RRID: CVCL_0045) (HEK-293 DKO)
and pa en al HEK-293 cells (RRID: CVCL_0045) we e kindly
supplied by Mohamed T ebak (Depa men o Pha macology
and Chemical Biology, Uni e si y o Pi sbu gh, Pi sbu gh,
PA). Cells we e cul u ed a 37Cwi ha5%CO
2
in high-glucose
Dulbecco’smodified Eagle’s medium (DMEM) supplemen ed
wi h 10% ( ol/ ol) e al bo ine se um and 100 U/mL penicillin
and s ep omycin, as p e iously desc ibed (30). Mycoplasma-
ee cul u es we e checked using con ocal mic oscopy. Fo
Wes e n blo ing assay, a ound 5 10
6
cells we e pla ed on
100-mm pe i dish, whe eas o Ca
2þ
imaging, eGFP fluo es-
cen epo e assay and con ocal mic oscopy cells (4 10
5
)
we e seeded in a 35-mm six-well mul idish. Fo ansien ans-
ec ions, cells we e g own o 60%–80% confluency and
ans ec ed wi h indica ed plasmids using Dha maFECT
kb ans ec ion eagen and we e used 24 h a e ans ec-
ion. Cell iabili y a e ans ec ion h oughou he s udy,
es ima ed using he Li e/Dead iabili y/cy o oxici y ki , was in
he ange be ween 92 and 95%. All p ocedu es we e app o ed
by he E hics Commi ee o Uni e si y o Ex emadu a and
Se icio Ex eme~
no de Salud.
Wes e n Blo ing
Wes e n blo ing was pe o med as desc ibed p e iously
(31). B iefly, cells we e lysed wi h ice-cold RIPA bu e
(150mMNaCl,25mMT is,5mMEDTA,1%T i onX-100,
1% sodium deoxychola e, 0.1% SDS; pH 7.6) supplemen ed
wi h comple e EDTA- ee p o ease inhibi o cock ail. Cell
lysa es we e homogenized using an ul asonic homoge-
nize Sonoplus HD 2200.2 (Bandelin elec onic GmbH &
Co, Be lin, Ge many) and subsequen ly cen i uged o
15 min a 16,000 gand 4C. La e , Laemmli sample bu e
[20% ( ol/ ol) glyce ol, 4% (w / ol) SDS, 160 mM T is-HCl
pH 6.8, 10% ( ol/ ol) 2-me cap oe hanol] we e added. Cell
lysa es we e esol ed by 12% SDS-PAGE, and sepa a ed
p o eins we e elec opho e ically ans e ed on o ni o-
cellulose memb anes o subsequen p obing. A e blocking
esidual p o ein binding si e wi h o e nigh incuba ion o he
blo s wi h 10% (w / ol) BSA in T is-bu e ed saline wi h 0.1%
Tween-20 (TBST). Immunode ec ion o O ai1, O ai2, O ai3,
STIM1, STIM2, FLNA, FLNA phospho yla ed a Se 2152, and
b-ac in was achie ed by incuba ion o 1 h wi h an i-O ai1,
an i-O ai3 o an i-phospho-FLNA (Se 2152) an ibody-dilu ed
1:1,000 in T is-bu e ed saline wi h Tween (TBST)-, o by incu-
ba ion o 1 h wi h an i-O ai2, an i-STIM1, an i-STIM2 o an i-
FLNA an ibody, -dilu ed 1:500 in TBST- o by incuba ion o 1
hwi han i-b-ac in an ibody-dilu ed 1:2,000 in TBST-. To
de ec he p ima y an ibody, blo s we e incuba ed o 1 h wi h
fluo escen goa an i- abbi IgGS a B igh Blue700an ibody
(RRID: AB_2721073) o goa an i-mouse IgG S a B igh Blue
700 dilu ed 1:3,000 in TBST. In addi ion, p ima y an ibodies
we e also de ec ed using ho se adish pe oxidase-conjuga ed
goa an i- abbi IgG an ibody o ho se adish pe oxidase-con-
juga ed goa an i-mouse IgG an ibody dilu ed 1:10,000 in
TBST and hen, in his case, blo s we e exposed o enhanced
chemiluminescence eagen s o 5 min. In bo h cases, he
an ibody binding was de ec ed wi h a ChemiDoc MP Imaging
Sys em (Bio-Rad Labo a o ies, Inc., He cules, CA) and he
densi y o bands was measu ed using Image Lab 6.1 So wa e
(Bio-Rad Labo a o ies, He cules, CA). Da a we e no malized
o he amoun o b-ac in om he same gel.
eGFP Fluo escen Repo e Assay
HEK-293 O ai1-KO non ans ec ed o ans ec ed wi h
pEGFP-N1-O ai1a-eGFP o pEGFP-N1-O ai1b-eGFP plasmids
we e s imula ed wi h 100 μg/mL cycloheximide (CHX) o 0,
6, and 9 h in he absence o p esence o 1 μMbafilomycin A1
(BFA) o 10 lM MG132. La e , cells we e lysed wi h ice-cold
NP-40 bu e (137 mM NaCl, 20 mM T is, 2 mM EDTA, 10%
glyce ol, 1% Nonide P-40, 1 mM Na
3
VO
4
; pH 8) supple-
men ed wi h comple e EDTA- ee p o ease inhibi o cock ail.
eGFP fluo escence was measu ed using he Va ioskan LUX
mic opla e mul imode eade (The mo Fishe Scien ific,
Wal ham, MA). The exci a ion/emission wa eleng hs in he
fluo escence assay we e 485 nm/518 nm. eGFP fluo escence
was no malized wi h he o al amoun o p o eins measu ed
wi h BCA p o ein assay ki .
De e mina ion o Cy osolic F ee-Ca
21
Concen a ion
Cells we e loaded wi h he Ca
2þ
fluo escen p obe u a-2 by
incuba ion wi h 2 μM u a-2/AM o 30 min a 37C. Cul u ed
cells on co e slips moun ed on a pe usion chambe we e
placed on he s age o an epifluo escence in e ed mic o-
scope (Nikon Eclipse Ti2, Ams e dam, The Ne he lands) wi h
an image acquisi ion and analysis sys em o ideomic o-
scopy (NIS-Elemen s Imaging So wa e .5.02.00, Nikon,
Ams e dam, The Ne he lands). Cells supe usion was ca ied
ou a oom empe a u e wi h HEPES-bu e ed saline (HBS)
con aining (in mM) 125 NaCl, 5 KCl, 1 MgCl
2
, 5 glucose, and 25
HEPES, pH 7.4, supplemen ed wi h 0.1% (w / ol) BSA. Cells
we e e alua ed a 40 magnifica ion (Nikon CFI S FLUOR
40Oil, Ams e dam, The Ne he lands) and we e al e na-
i ely exci ed wi h ligh om a xenon lamp passed h ough
a high-speed monoch oma o Op oscan ELE 450 (Cai n
Resea ch, Fa e sham, UK) a 340/380 nm. Fluo escence
emission was de ec ed a 510 nm using a cooled digi al
sCMOS came a PCO Panda 4.2 (Exceli as PCO GmbH,
Ge many) and eco ded using NIS-Elemen s AR so wa e
(Nikon, Ams e dam, The Ne he lands). Fluo escence a io
(F340/F380) was calcula ed pixel by pixel, and he da a we e
p esen ed as DF
340
/F
380
as desc ibed p e iously (18). TG-e oked
Ca
2þ
elease and Ca
2þ
en y we e es ima ed as he a ea unde
he cu e measu ed as he in eg al o he ise in u a-2 fluo es-
cence a io 4 min a e he addi ion o TG ( o Ca
2þ
elease) o
Ca
2þ
( o Ca
2þ
en y), espec i ely, and aking a sample e e y
second. Al e na i ely, Ca
2þ
en y was de e mined as he ini ial
peak in u a-2 340/380 fluo escence a io abo e basal le els a -
e e-addi ion o Ca
2þ
o he ex acellula medium.
FLNA CLEAVING MODULATES O ai1 EXPRESSION
AJP-Cell Physiol doi:10.1152/ajpcell.00745.2024 www.ajpcell.o g C659
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Con ocal Mic oscopy
The subcellula loca ion o DsRed-FLNA
16–24
,STIM1-
mChe y, o O ai1-eGFP was de e mined by imaging cells
24 h pos ans ec ion, upon 30-min incuba ion a 37C
wi h o wi hou Hoechs 33258 (1 μg/mL) o nuclea s ain-
ing. The imaging was pe o med using a con ocal mic o-
scope (LSM900, Ca l Zeiss, Ge many) equipped wi h a 63
FLNA CLEAVING MODULATES O ai1 EXPRESSION
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oil imme sion objec i e and an image acquisi ion and anal-
ysis sys em o ideo mic oscopy (ZenBlue 3.4 So wa e,
Ca l Zeiss, Ge many). To assess he exp ession o DsRed-
FLNA
16–24
, se en o en andom egions o in e es (ROIs),
each measu ing 2.5 lm
2
, we e selec ed o fluo escence
measu emen , co ec ed by backg ound sub ac ion and
h esholding me hods, and conduc ed using a cus om
sc ip in eg a ed in o he FIJI ImageJ 1.54 so wa e pla -
o m (RRID:SCR_003070).
Bio inyla ion o Cell Su ace P o eins
Labeling and isola ion o plasma memb ane p o eins we e
pe o med by su ace bio inyla ion assay, as desc ibed p e i-
ously (18). HEK-293 and O ai1-KO HEK-293 cells we e
washed h ee imes wi h phospha e-bu e ed saline (PBS,
NaCl 137 mM, KCl 2.7 mM, KH
2
PO
4
, 1.5 mM, Na
2
HPO
4
·2H
2
O
8 mM, pH 8). Cells we e hen incuba ed a 4C o 1 h wi h
bio ynila ion bu e (PBS supplemen ed wi h 1 mg/mL EZ-
Link sul o-NHS-LC-bio in). The eac ion was e mina ed by
addi ion o T is base (final concen a ion 50 mM). Following
bio inyla ion, cells we e washed wice in PBS, dis up ed
using Nonide P-40 bu e and sonica ed. Cell lysa es we e
cen i uged (16,000 g o 5 min a 4C), and p o ein concen-
a ion was measu ed using BCA assay. Samples we e incu-
ba ed wi h 50 lL s ep a idin beads a 4C o 2 h and
esuspended in Laemmli bu e o subsequen analysis by
Wes e n blo ing.
S a is ical Analysis
All expe imen s we e pe o med and analyzed using s a -
egies o a oid bias. Da a a e p esen ed as he means ± SE.
Analysis o s a is ical significance was pe o med using
G aphPad P ism .8.4.3 (RRID:SCR_002798, G aphPad
So wa e, San Diego, CA). K uskal–Wallis es combined
wi h Dunn’s pos hoc es we e used o compa e he di e -
en expe imen al g oups. Fo compa ison be ween wo
g oups, he Mann–Whi ney U es was used. All da a wi h
P<0.05 was deemed significan .
RESULTS
Exp ession o O ai and STIM Membe s and Filamin A in
Colo ec al Adenoca cinoma Cell Lines and No mal
Colon Mucosa NCM460 Cells
Se e al s udies ha e e ealed ha he exp ession o O ai
and STIM p o eins is significan ly al e ed in colo ec al ade-
noca cinoma HT-29 cells as compa ed wi h no mal colon
mucosa NCM460 cells, leading o enhanced SOCE accompa-
nied by a educed abili y o accumula e Ca
2þ
in o he in a-
cellula s o es (29,30,32). Acco ding o his, fi s o all, we
ha e u he analyzed SOCE as well as he exp ession o he
key SOCE molecula playe s in HT-29 cells, he mos widely
s udied colo ec al adenoca cinoma cell line, in compa ison
wi h no mal colon mucosa cells. As shown in Fig. 1A, ea -
men o NCM460 cells wi h TG in he absence o ex acellu-
la Ca
2þ
esul s in a ansien inc ease in cy osolic ee-Ca
2þ
concen a ion [Ca
2þ
]
i
, as a esul o passi e Ca
2þ
e flux om
he in acellula s o es as a esul o SERCA inhibi ion.
Subsequen addi ion o 1.8 mM Ca
2þ
o he ex acellula me-
dium led o a mo e sus ained inc ease in [Ca
2þ
]
i
, which is in-
dica i e o SOCE. In colo ec al adenoca cinoma HT-29 cells,
TG-e oked Ca
2þ
e flux om he in acellula Ca
2þ
s o es was
significan ly a enua ed and SOCE was ound o be signifi-
can ly enhanced as compa ed wi h ha obse ed NCM460
(Fig. 1, A–D,P<0.0001). The analysis o he exp ession o O ai
and STIM p o eins e ealed ha , in ag eemen wi h p e ious
s udies (29), HT-29 cells exhibi a significan ly g ea e exp es-
sion o O ai1, pa icula ly O ai1a,O ai3,andSTIM1(Fig. 1, E–
H;P<0.05). Fu he mo e, we ound ha STIM2 is o e ex-
p essed in HT-29 cells as compa ed wi h NCM460 cells (Fig. 1I;
P<0.001), an obse a ion ha we ha e ecen ly epo ed (30).
The e o e, ou cu en esul s confi m p e ious obse a ions.
FLNA is a cy oskele al p o ein ha plays a ele an ole
in he modula ion o SOCE (20). Da a om he Clinical
P o eomic Tumo Analysis Conso ium (33) (CPTAC) (h ps://
ualcan.pa h.uab.edu) ha e shown ha FLNA exp ession a
he p o ein le el is educed in a numbe o cance ypes
Figu e 1. S o e-ope a ed Ca
2þ
en y and exp ession o O ai, STIM, and FLNA in colo ec al cance and no mal mucosa cells. A: u a-2-loaded no mal mu-
cosa NCM460 cells and colo ec al cance HT-29 cells we e suspended in a Ca
2þ
- ee (100 lM EGTA) HBS and hen s imula ed wi h 2 lMTG ollowed
by ein oduc ion o ex e nal Ca
2þ
(final concen a ion 1.8 mM) o ini ia e Ca
2þ
en y. B–D: sca e plo s ep esen quan ifica ion o TG-e oked Ca
2þ
elease (B) and en y de e mined as he a ea unde he cu e (AUC; C)andCa
2þ
en y de e mined as he ini ial peak in u a-2 340/380 fluo escence a-
io abo e basal le els a e addi ion o Ca
2þ
o he ex acellula medium (D), de e mined as desc ibed in MATERIALS AND METHODS. Da a a e p esen ed as
means ± SE and a e s a is ically analyzed using Mann–Whi ney U es . P<0.0001 as compa ed wi h NCM460 cells. E–I: NCM460 and HT-29 cells
we e lysed. Cell lysa es we e ea ed in he absence (E,G–I) o he p esence o PNGase F (F) and we e hen subjec ed o 10% SDS-PAGE and Wes e n
blo ing wi h he an i-O ai1 (Eand F), an i-O ai3 (G), an i-STIM1 (H) o an i-STIM2 (I) an ibody. J: NCM460, HT-29, Caco-2 and HCT116 cells we e lysed and
hen subjec ed o 10% SDS-PAGE and Wes e n blo ing wi h he an i-FLNA, an i-STIM1, and an i-O ai1 (J) an ibody, as desc ibed in MATERIALS AND
METHODS.E–J: memb anes we e ep obed wi h he an i-b-ac in an ibody o p o ein loading con ol. Molecula masses indica ed on he igh we e de e -
mined using molecula -mass ma ke s un in he same gel. Blo s a e ep esen a i e o h ee o ou sepa a e expe imen s. Ba g aphs ep esen he quan-
ifica ion o he p o ein exp ession as old inc ease o e he le el in NCM460 cells, p esen ed as means ± SE. Da a we e s a is ically analyzed using
Mann–Whi ney U es [excep o J, whe e K uskal–Wallis es wi h mul iple compa isons (Dunn’s es )wasused].P<0.05, P<0.01, P<0.001,
and P<0.0001 as compa ed wi h NCM460 cells. #P<0.05, ###P<0.001, and ####P<0.0001 as compa ed wi h HT-29 cells. K–O: HT-29 cells
we e ea ed wi h 1 lM calpep in o 24 h o he ehicle as con ol. Cells we e hen lysed and subjec ed o 10% SDS-PAGE and Wes e n blo ing wi h he
an i-FLNA (K), an i-O ai1 (L), an i-O ai3 (M), an i-STIM1 (N), and an i-STIM2 (O) an ibody, as desc ibed in MATERIALS AND METHODS. Memb anes we e e-
p obed wi h he an i-b-ac in an ibody o p o ein loading con ol. Molecula masses indica ed on he igh we e de e mined using molecula -mass
ma ke s un in he same gel. Blo s a e ep esen a i e o ou sepa a e expe imen s. Ba g aphs ep esen he quan ifica ion o p o ein exp ession as old
inc ease o e he le el in he absence o calpep in and p esen ed as means ± SE. Da a we e s a is ically analyzed using Mann–Whi ney U es . P<
0.001 and P<0.0001. P–S: HT-29 cells we e ea ed wi h 1 lM calpep in o 24 h o he ehicle as con ol. Cells we e loaded wi h u a-2, suspended
in a Ca
2þ
- ee (100 lM EGTA) HBS, and hen s imula ed wi h 2 lMTG ollowedby ein oduc iono ex e nalCa
2þ
(final concen a ion 1.8 mM) o ini ia e
Ca
2þ
en y. Q–S: sca e plo s ep esen quan ifica ion o TG-e oked Ca
2þ
elease (Q)Ca
2þ
en y de e mined as he a ea unde he cu e (AUC; R)
and Ca
2þ
en y de e mined as he ini ial peak in u a-2 340/380 fluo escence a io abo e basal le els a e addi ion o Ca
2þ
o he ex acellula medium
(S), as desc ibed in MATERIALS AND METHODS. Da a a e p esen ed as means ± SE and a e s a is ically analyzed using Mann–Whi ney U es . P<0.01 and
P<0.0001. FLNA, Filamin A; HBS, HEPES-bu e ed saline; TG, Thapsiga gin.
FLNA CLEAVING MODULATES O ai1 EXPRESSION
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including colon, o a ian and lung cance , which exhibi
g ea e SOCE and O ai1 and STIM1 exp ession (29,34–36).
Conce ning colon cance , a significan ly educed FLNA p o-
ein le el has been de ec ed in pa ien s samples compa ed
wi h no mal colon issue (Supplemen al Fig. S1). Hence, we
ha e explo ed he possible di e ences in he exp ession o
his p o ein in he adenoca cinoma cell lines HT-29, Caco-2,
and HCT116, as well as in no mal colon mucosa NCM460
cells. As shown in Fig. 1J, Wes e n blo ing o whole cell
lysa es wi h a specific an i-FLNA an ibody e ealed a band
ha co esponds o he p edic ed size o ull-leng h FLNA
(280 kDa). In addi ion o he 280-kDa band, we de ec ed an
190 kDa agmen co esponding o he la ge p oduc o
FLNA clea age by calpain (37). In e es ingly, we ound ha
he exp ession o ull-leng h FLNA was significan ly educed
in HT-29 cells as compa ed wi h he colo ec al cell lines
Caco-2 and HCT116 o NCM460 cells (Fig. 1J;P<0.0001).
Recip ocally, he exp ession o he 190 kDa FLNA ag-
men was enhanced in HT-29 cells and, o a lesse ex en ,
in NCM460 cells (Fig. 1J;P<0.0001). These obse a ions
s ongly sugges ha FLNA is mos ly clea ed in colo ec al
adenoca cinoma HT-29 cells as compa ed wi h Caco-2 o
HCT116 cells o he no mal colon mucosa cells. As shown
in Fig. 1J, HT-29 cells show enhanced O ai1 and STIM1
exp ession as compa ed wi h he colo ec al cell lines
Caco-2 and HCT116 o NCM460 cells; howe e , he mecha-
nism unde lying he inc ease in O ai1 and STIM1 p o ein
le el emains unknown.
Ou esul s indica e ha FLNA is mos ly clea ed in HT-29
cells leading o a agmen ha esembles he p e iously
men ioned p oduc o calpain p o eolysis, which is in ol ed
in ansc ip ional egula ion (38). To figu e ou whe he
FLNA is clea ed by calpain in HT-29 cells and he possi-
ble oleo FLNAclea ageinO ai1andSTIM1p o eincon-
en , cells we e ea ed wi h he cell-pe mean calpain
inhibi o calpep in. As shown in Fig. 1K, ea men o HT-
29 cells wi h 1 lMcalpep in o 24hsignifican ly educed
FLNA clea age as de ec ed by he enhancemen o he
exp ession o ull-leng h FLNA and a enua ion o he
FLNA agmen , which s ongly sugges s ha FLNA is
clea ed by calpain. Fu he mo e, ea men wi h calpep-
in significan ly educed O ai1 and STIM1 exp ession
wi hou ha ing any e ec on he p o ein con en o O ai3
and STIM2 (Fig. 1, L–O;P<0.001), consequen ly, SOCE
was significan ly educed in cells ea ed wi h calpep in
(Fig. 1, P–S;P<0.01). We u he ound ha calpep in did
no induce a de ec able educ ion in he abili y o STIM1
o o m clus e s (Supplemen al Fig. S2). These findings
indica e ha calpain is essen ial o FLNA clea age and
ha his e en modula es O ai1 and STIM1 p o ein
exp ession.
FLNA phospho yla ion a Se 2152 has been epo ed o
be essen ial o i s biological unc ions (23,39), including
he modula ion o SOCE (20). Wes e n blo ing o whole
cell lysa es wi h a specific an i-phospho-FLNA (FLNA
P-Se 2152) an ibody de ec ed a single band a 280 kDa
co esponding o he ull-leng h FLNA ha was signifi-
can ly educed in HT-29 cell lysa es, p obably as a esul o
he educed exp ession o ull-leng h FLNA in hese cells
(Supplemen al Fig. S3, Aand B;P<0.05). Fu he mo e,
no maliza ion o he amoun o pFLNA o he o al FLNA
e ealed ha ela i e FLNA phospho yla ion a Se 2152 is
significan ly g ea e in HT-29 cells han in NCM460 cells.
We we e unable o de ec phospho yla ion o he FLNA
agmen s. We ha e u he in es iga ed he unc ional
ole o FLNA se ine phospho yla ion in he egula ion o
SOCE in HT-29 by ansien ly exp essing wild- ype FLNA
o he nonphospho yla able FLNA S2152A mu an . As
shown in Supplemen al Fig. S3, exp ession o wild- ype
FLNA in HT-29 cells significan ly educed SOCE and
enhanced TG-e oked Ca
2þ
elease om he in acellula
s o es (Supplemen al Fig. S3, C–F;P<0.0001). By con-
as , exp ession o he nonphospho yla able FLNA mu-
an was wi hou e ec on TG-induced Ca
2þ
elease o
SOCE (Supplemen al Fig. S3, C–F), which suppo s a ole
o FLNA Se 2152 phospho yla ion in he modula ion o
hese e en s. In addi ion, ou esul s indica e ha
exp ession o wild- ype FLNA significan ly a enua ed
O ai1 and STIM1 exp ession (Supplemen al Fig. S3, G–J;
P<0.05). Meanwhile, he exp ession o he FLNA S2152A
mu an educed STIM1 exp ession wi hou ha ing any
significan e ec on he p o ein con en o O ai1, O ai3,
o STIM2 as compa ed wi h non ans ec ed HT-29 cells
(Supplemen al Fig. S3, G–J;P<0.05). Howe e , i is
wo h men ioning ha O ai1 exp ession in cells exp ess-
ing wild- ype FLNA o he FLNA S2152A mu an was no
significan ly di e en (Supplemen al Fig. S3G;P¼
0.537). These obse a ions indica e ha FLNA phospho-
yla ion a Se 2152 plays a significan unc ional ole in
he modula ion o SOCE and he accumula ion o Ca
2þ
in o he in acellula s o es in he colo ec al adenoca ci-
noma cell line HT-29, as p e iously epo ed (20).
The FLNA 16–24 F agmen Enhances O ai1 and STIM1
P o ein Con en
Clea age o FLNA a he fi s clea ing si e leads o he o -
ma ion o 190- and 90-kDa agmen s, co esponding
o he N- e minal 15 epea s o FLNA and he C- e minal
egion encompassing epea s 16–24 (FLNA
16–24
)(38,40). The
FLNA
16–24
agmen has been epo ed o modula e gene
ansc ip ion by ep essing and ogen ecep o ac i i y
(38,41). Hence, we ha e u he in es iga ed he possible
ole o he FLNA
16–24
agmen on he exp ession o O ai
and STIM p o eins by ex ending ou s udies in HEK-293
cells, a commonly used cell model o cell biology, whe e
FLNA is no na u ally clea ed (Fig. 2A). To explo e whe he
he FLNA
16–24
agmen is able o egula e O ai1 and STIM1
p o ein con en , HEK-293 cells we e ans ec ed wi h DsRed-
FLNA
16–24
exp ession plasmid and he exp ession o O ai
and STIM p o eins, as well as SOCE, was analyzed. As
shown in Fig. 2A, Wes e n blo ing o HEK-293 lysa es wi h
an i-FLNA an ibody e ealed a single band o 280-kDa
co esponding o he na i e, ull-leng h, FLNA in HEK-293
cells ans ec ed wi h emp y ec o and wo bands o
280- and 120-kDa in HEK-293 cells ans ec ed wi h
DsRed-FLNA
16–24
exp ession plasmid, he la e co e-
sponding wi h he p edic ed size o DsRed-FLNA
16–24
ag-
men . Exp ession o DsRed-FLNA
16–24
was u he confi med
by con ocal mic oscopy, which e ealed a cy oplasmic loca ion
o he FLNA agmen (Fig. 2A). Exp ession o he FLNA
16–24
agmen in HEK-293 cells significan ly enhanced he p o ein
FLNA CLEAVING MODULATES O ai1 EXPRESSION
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Figu e 2. Exp ession o he FLNA
16–24
cons uc enhances he p o ein le el o O ai1 and STIM1 in HEK-293 cells. A: HEK-293 cells we e ans ec ed wi h
DsRed-FLNA
16–24
agmen o emp y ec o and 48 h la e cells we e ei he lysed o isualized by con ocal mic oscopy. Top: cell lysa es we e subjec ed
o 10% SDS-PAGE and Wes e n blo ing wi h he an i-FLNA an ibody, as desc ibed in MATERIALS AND METHODS. Molecula masses indica ed on he igh
we e de e mined using molecula -mass ma ke s un in he same gel. Blo s a e ep esen a i e o ou sepa a e expe imen s. Bo om:DsRedfluo escence
was de ec ed using an LSM900 con ocal mic oscope. The images show ep esen a i e con ocal images o DsRed-FLNA
16–24
and nuclea s aining wi h
Hoechs 33342. The scale ba ep esen s 10 μm. B–E: HEK-293 cells we e ans ec ed wi h DsRed-FLNA
16–24
agmen o emp y ec o , as desc ibed.
Fo y-eigh hou s la e , cells we e lysed and subjec ed o 10% SDS-PAGE and Wes e n blo ing wi h he an i-O ai1 (B), an i-O ai3 (C), an i-STIM1 (D), and
an i-STIM2 (E) an ibody. Memb anes we e ep obed wi h he an i-b-ac in an ibody o p o ein loading con ol. Molecula masses indica ed on he igh
we e de e mined using molecula -mass ma ke s un in he same gel. Blo s a e ep esen a i e o ou sepa a e expe imen s. Ba g aphs ep esen he
quan ifica ion o p o ein exp ession as old inc ease o e he le el in mock- ans ec ed cells and p esen ed as means ± SE. Da a we e s a is ically ana-
lyzed using Mann–Whi ney U es . P<0.05 and P<0.01. F–I: HEK-293 cells we e ans ec ed wi h DsRed-FLNA
16–24
agmen o emp y ec o . Fo y-
eigh hou s la e , cells we e loaded wi h u a-2. Cells we e hen suspended in a Ca
2þ
- ee (100 lM EGTA) HBS and s imula ed wi h 2 lM TG ollowed by
ein oduc ion o ex e nal Ca
2þ
(final concen a ion 1.8 mM) o ini ia e Ca
2þ
en y. G–I: sca e plo s ep esen quan ifica ion o TG-e oked Ca
2þ
elease
(G)Ca
2þ
en y de e mined as he a ea unde he cu e (AUC; H)andCa
2þ
en y de e mined as he ini ial peak in u a-2 340/380 fluo escence a io
abo e basal le els a e addi ion o Ca
2þ
o he ex acellula medium (I), as desc ibed in MATERIALS AND METHODS.Da aa ep esen edasmeans±SEand
a e s a is ically analyzed using Mann–Whi ney U es . P<0.05 and P<0.01. FLNA, Filamin A; HBS, HEPES-bu e ed saline; TG, Thapsiga gin.
FLNA CLEAVING MODULATES O ai1 EXPRESSION
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con en o O ai1 and STIM1, wi hou ha ing any e ec on he
exp ession o O ai3 and STIM2 (Fig. 2, B–E;P<0.05). As a
esul , he exp ession o he FLNA
16–24
agmen signifi-
can ly enhanced SOCE and educed he abili y o HEK-
293 cells o accumula e Ca
2þ
in o he in acellula s o es
(Fig. 2, F–I;P<0.05). These findings a e consis en wi h
he e ec o calpep in on O ai1 and STIM1 exp ession in
HT-29 cells and esembles he pheno ype (enhanced O ai1
and STIM1 p o ein con en and SOCE) o he HT-29 cell
line.
The FLNA 16–24 F agmen Enhances O ai1 and STIM1
P o ein Con en by A enua ing Deg ada ion
The p o ein con en is he esul o a balance be ween
p o ein syn hesis and deg ada ion. We ha e u he
explo ed he mechanism unde lying he egula ion o
O ai1 and STIM1 p o ein con en by he FLNA
16–24
ag-
men by using he p o ein syn hesis inhibi o cyclohexi-
mide (CHX). HEK-293 cells we e ans ec ed wi h DsRed-
FLNA
16–24
agmen exp ession plasmid o emp y ec o
and we e u he ea ed wi h CHX o he ehicle o 9 h.
As depic ed in Fig. 3, O ai1 and STIM1 p o ein exp ession
inc eases by 40 and 30%, espec i ely, a e ans ec ion
o he FLNA agmen (P<0.01). CHX by i sel a enua ed
he exp ession o O ai1 and STIM1 p o eins by 38 and
35%, espec i ely (Fig. 3). In he p esence o CHX, he
exp ession o O ai1 and STIM1 inc eases a e he exp es-
sion o he FLNA agmen by 40 and 37%, espec i ely,
(Fig. 3;P<0.01). The e o e, ou esul s indica e ha he
FLNA
16–24
agmen was able o enhance O ai1 and STIM1
exp ession in he absence o p o ein syn hesis, which
s ongly sugges ha he e ec is likely o be media ed
by an inc ease in p o ein hal -li e induced by inhibi ion
o p o ein deg ada ion.
The O ai1 Va ian s O ai1aand O ai1bExhibi Diffe en
Deg ada ion Ra es
As he FLNA
16–24
agmen modula es he deg ada ion o
O ai1 and wo O ai1 a ian s ha e been iden ified, we ha e
u he analyzed he e ec o he FLNA
16–24
agmen on he
deg ada ion o O ai1aand O ai1b.Fi s ,weha eassessed he
deg ada ion o bo h O ai1 a ian s in O ai1-KO HEK-293
ans ec ed wi h O ai1ao O ai1bby es ima ing hei p o ein
con en in he p esence o he p o ein syn hesis inhibi o
CHX. O ai1-KO HEK-293 was ans ec ed wi h cy omegalo i-
us (CMV)-d i en O ai1a-eGFP o O ai1b-eGFP, and he p o-
ein con en o he O ai1 a ian s was de e mined by
Wes e n blo ing using an an i-O ai1 an ibody. As depic ed
in Fig. 4A,O ai1ap o ein con en dec eases by 20 and 30%
a e cell exposu e o CHX o 6 and 9 h, espec i ely (P<
0.01). By con as , he p o ein con en o O ai1bwas una -
ec ed by inhibi ion o p o ein syn hesis a leas du ing 9 h.
Simila esul s we e ob ained when he p o ein con en o
O ai1a-eGFP o O ai1b-eGFP was de e mined by eGFP fluo-
escence quan ifica ion (Fig. 4B). These findings p o ide o
he fi s ime e idence suppo ing ha he s abili y o O ai1b
is g ea e han ha o O ai1a.
In mammalian cells, p o ein deg ada ion mainly occu s
h ough he ubiqui in-p o easome pa hway and he lysoso-
mal-dependen p o eolysis. To elucida e he pa hway in ol ed
in O ai1 deg ada ion, cells we e ea ed wi h bafilomycin A1
(BFA), a selec i e inhibi o o acuola H
þ
-ATPase ha p e-
en s lysosomal deg ada ion (42)o MG132,aninhibi o
o p o easome (43), and O ai1 a ian p o ein con en was
Figu e 3. Exp ession o he FLNA
16–24
cons uc a enua es O ai1 and STIM1 deg ada ion. HEK-293 cells we e ans ec ed wi h DsRed-FLNA
16–24
ag-
men o emp y ec o , as indica ed. Fo y-eigh hou s la e , cells we e ei he ea ed wi h cycloheximide (CHX; 100 lg/mL) o he ehicle o 9 h and
lysed. Cell lysa es we e subjec ed o 10% SDS-PAGE and Wes e n blo ing wi h he an i-FLNA an ibody and ei he an i-O ai1 (A)o an i-STIM1(B)an ibody.
Memb anes we e ep obed wi h he an i-b-ac in an ibody o p o ein loading con ol. Molecula masses indica ed on he igh we e de e mined using
molecula -mass ma ke s un in he same gel. Blo s a e ep esen a i e o ou sepa a e expe imen s. Ba g aphs ep esen he quan ifica ion o p o ein
exp ession as old inc ease o e he le el in mock- ans ec ed cells no ea ed wi h CHX. Da a a e p esen ed as means ± SE and s a is ically analyzed
using K uskal–Wallis es wi h mul iple compa isons (Dunn’s es ).P<0.01 and P<0.001 as compa ed o mock- ans ec ed cells no ea ed wi h
CHX. ##P<0.01 as compa ed wi h DsRed-FLNA
16–24
agmen - ans ec ed cells no ea ed wi h CHX. CHX, cycloheximide; FLNA, Filamin A.
FLNA CLEAVING MODULATES O ai1 EXPRESSION
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de e mined by he de e mina ion o eGFP fluo escence. As
epo ed abo e, ea men o 9 h wi h CHX by i sel sig-
nifican ly educed O ai1aexp ession (Fig. 4C;P<0.001).
In he p esence o CHX and BFA, he O ai1ap o ein con-
en was simila o ha in cells no ea ed wi h CHX
(Fig. 4C), indica ing ha BFA impai s O ai1ap o ein deg-
ada ion. By con as , MG132 was unable o e e se he
d op in O ai1ap o ein con en obse ed upon blockade o
p o ein syn hesis wi h CHX (Fig. 4C;P<0.01). The
exp ession o O ai1bwas una ec ed by ea men wi h
CHX in he absence o p esence o BFA and MG132
(Fig. 4C). These findings indica e ha O ai1adeg ada ion
occu s h ough lysosomal p o eolysis, which is consis en
wi h p e ious s udies by Yeh e al. (44).
The FLNA 16–24 F agmen A enua es O ai1aP o ein
Deg ada ion Re aining he Channel in he Plasma
Memb ane
We ha e specifically explo ed whe he he FLNA
16–24
ag-
men modifies he p o ein deg ada ion o O ai1ao O ai1b.
O ai1-KO HEK-293 cells we e ans ec ed wi h O ai1a-eGFP o
O ai1b-eGFP in he absence o p esence o DsRed-FLNA
16–24
agmen exp ession plasmid o emp y ec o and we e u -
he ea ed wi h CHX o he ehicle o 9 h. As depic ed in
Supplemen al Fig. S4 o na i e O ai1, de ec ion o O ai1 a -
ian s used o eGFP leads o se e al di use bands, as p e i-
ously desc ibed (16), likely due o N-linked glycosyla ion o
O ai1 (Fig. 5). As shown in Fig. 5,O ai1ap o einexp essionsig-
nifican ly inc eases a e ans ec ion o he FLNA agmen
(P<0.01). CHX by i sel significan ly a enua ed he exp es-
sion o O ai1a(P<0.001). In he p esence o CHX, he exp es-
sion o O ai1asignifican ly inc eases a e exp ession o he
FLNA agmen (Fig. 5;P<0.01) eaching a alue ha was
compa able o ha obse ed in un ea ed O ai1-KO HEK-293
cells exp essing O ai1a, which indica es ha he FLNA
16–24
agmen impai s O ai1ap o ein deg ada ion. Conce ning
O ai1b, nei he ea men wi h CHX no ans ec ion o he
DsRed-FLNA
16–24
agmen al e ed he p o ein exp ession a
leas a he ime in es iga ed, which is consis en wi h he low
a e o O ai1bp o ein deg ada ion.
We u he explo ed whe he he C- e minal agmen o
FLNA impai s O ai1adeg ada ion by impai ing p o ein
endocy osis by analyzing he exp ession o O ai1ain he
plasma memb ane by su ace p o ein bio inyla ion. HEK-
293 cells we e ans ec ed wi h DsRed-FLNA
16–24
agmen
exp ession plasmid o emp y ec o and, a e bio inyla ion,
Figu e 4. Analysis o O ai1aand O ai1bdeg ada ion. A:O ai1-KOHEK-293
cells we e ans ec ed wi h CMV-d i en O ai1a-eGFP (lanes 1–3)o O ai1b-
eGFP (lanes 4–6). Fo y-eigh hou s la e , cells we e hen ea ed in he
absence o p esence o cycloheximide (CHX; 100 lg/mL) o 6 o 9 h, as
indica ed, and lysed. Cell lysa es we e hen subjec ed o 10% SDS-PAGE
and Wes e n blo ing wi h an i-O ai1 an ibody, as desc ibed in MATERIALS
AND METHODS. Memb anes we e ep obed wi h he an i-b-ac in an ibody
o p o ein loading con ol. Molecula masses indica ed on he igh we e
de e mined using molecula -mass ma ke s un in he same gel. Blo s a e
ep esen a i e o h ee sepa a e expe imen s. Sca e plo s ep esen
O ai1a-eGFP and O ai1b-eGFP exp ession a he di e en expe imen al
condi ions. Da a a e p esen ed as means ± SE and s a is ically analyzed
using K uskal–Wallis es wi h mul iple compa isons (Dunn’s es ).P<
0.01 as compa ed wi h cells no ea ed wi h CHX. B:O ai1-KOHEK-293
cells we e ans ec ed wi h CMV-d i en O ai1a-eGFP o O ai1b-eGFP.
Fo y-eigh hou s la e , cells we e hen ea ed in he absence o p esence
o cycloheximide (CHX; 100 lg/mL) o 6 o 9 h, as indica ed and GFP fluo-
escence was de e mined as desc ibed in MATERIALS AND METHODS.Sca e
plo s ep esen O ai1a-eGFP and O ai1b-eGFP fluo escence a he di e -
en expe imen al condi ions. Da a a e p esen ed as means ± SE and s a is-
ically analyzed using K uskal–Wallis es wi h mul iple compa isons
(Dunn’s es ).P<0.01 as compa ed wi h cells no ea ed wi h CHX. C:
O ai1-KO HEK-293 cells we e ans ec ed wi h CMV-d i en O ai1a-eGFP o
O ai1b-eGFP, as indica ed. Fo y-eigh hou s la e , cells we e hen ea ed
in he absence o p esence o cycloheximide (CHX; 100 lg/mL) o 9 h,
alone o in combina ion wi h 1 lMbafilomycin A1 (BFA) o 10 lMMG132,as
indica ed, and GFP fluo escence was de e mined as desc ibed in
MATERIALS AND METHODS. Sca e plo s ep esen eGFP fluo escence a he
di e en expe imen al condi ions. Da a a e p esen ed as means ± SE and
s a is ically analyzed using K uskal–Wallis es wi h mul iple compa isons
(Dunn’s es ).P<0.01 and P<0.001 as compa ed wi h cells no
ea ed wi h CHX. ##P<0.01 and ###P<0.001 as compa ed wi h cells
ea ed wi h CHX and BFA. CHX, cycloheximide; KO, knockou .
FLNA CLEAVING MODULATES O ai1 EXPRESSION
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