5086 Author Correction

Mechanism of polarized lysosome exocytosis in epithelial cells Jin Xu, Kimberly A. Toops, Fernando Diaz, Jose Maria Carvajal-Gonzalez, Diego Gravotta, Francesca Mazzoni, Ryan Schreiner, Enrique Rodriguez-Boulan and Aparna Lakkaraju

Journal of Cell Science 126, 5086 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.143982

There was an error published in J. Cell Sci. 125, 5937–5943.

In Fig. 4B the same image was inadvertently presented as the xy section (top panels) of syntaxin 4 staining in MDCK wild-type and MDCK m1A-KD cells. In the correct version (shown below), the appropriate xy image for m1A-KD (top panel, middle) matches the corresponding xz panel (bottom panel) that was part of the original figure.

The mistake in the figure did not affect the conclusions of the paper.

The correct Fig. 4B is shown below.

The authors apologise for this mistake. Journal of Cell Science o:10.1242/jcs.109421 doi: 5937–5943 125, ß Science Cell of Journal 2012 September 17 Accepted yatctasiso L ta. 08 hn ta. 2007). al., synaptotagmin sensor et calcium lysosome Zhang lysosomal calcium-induced the 2008; include in al., exocytosis implicated et machinery Molecular (Li transmission synaptic USA rnec Mazzoni Francesca xouet acu oohrsadpr-omn toxins Rodrı pore-forming 2002; al., and et important Jaiswal ionophores 2009; an al., calcium after et as repair (Divangahi membrane to emerged as such exposure since paradigms diverse plasma has in mechanism the 2002). exocytosis of and (Andrews, parasite region influx Lysosome invading the calcium the surrounds with triggers that lysosomes membrane membrane of cell fusion the subsequent to parasite of the invasion cell during observed 97 n o rpgtn h Ca the propagating for and 1997) i Xu Jin in cells exocytosis epithelial lysosome polarized of Mechanism Report Short rnin nraei nrclua acu ([Ca calcium a intracellular to in response in al., increase et lysosomes (Stinchcombe transient ‘conventional’ apoptosis of cell Exocytosis target In 2004). effect delivers proteins. lysosome- to synapse granules of immunological lytic or the repertoires at defined secretion lymphocytes lysosomes lysosome store CTLs, T secretory that organelles cytotoxic specialized terminal related and have than as al., platelets (CTLs) recognized et more (Luzio Melanocytes, now homeostasis little are cellular 2007). maintaining cell, be in players the central to of compartments thought degradative long Lysosomes, Introduction and process. this apical of both regulator crucial and a MDCK, is involved wild-type AP-1 machinery molecular that words: in the suggest Key into exocytosis and insight fundamental epithelia basolateral with provide polarized Consistent results inhibits membranes. in Our basolateral repair AP-1B. 4 and or membrane apical epithelial- AP-1A syntaxin in the the lacking both or of cells AP-1A with in ubiquitous fuse Overloading depletion exocytosis the to basolateral exocytosis. either RNAi-mediated lysosomes lacking to causes cells findings, This barrier AP-1 In non-polar. adaptor domain. a these is basolateral clathrin is 4 the The but polarized syntaxin to traffic. actin actin, cells, 4 AP-1B, lysosomal This cortical syntaxin of specific paralyzes (MDCK) targeting membrane. that Depolymerization cholesterol accurately kidney excess AP-1. hypothesis basolateral for external that adaptor canine responsible the suggesting the the is clathrin exocytosis, Madin-Darby supporting with the inhibits and fusion, polarized cholesterol and predominantly organism lysosome with in cholesterol lysosomes fuse apical the membrane that causes cytoskeleton, to between find actin microtubules, lysosomes barrier We the not cause by pathogens. main we regulated toxins Here, against the is membranes. pore-forming defense epithelia, exocytosis torn of or pathogen of polarized limiting line ionophores resealing in repair, rapid first calcium membrane exocytosis facilitates the for exocytosis lysosome crucial and lysosome of is environment cells, that mechanism non-polarized process In the calcium-dependent debris. a investigate cellular is clearing membrane plasma and the entry with lysosomes of Fusion Summary ( correspondence for *Authors 3 2 1 eateto eladDvlpetlBooy el eia olg fCrelUiest,NwYr,N 02,USA 10021, NY York, New University, USA Cornell 10021, of NY York, College New Medical University, 53705, Cornell Weill of WI Biology, College Madison, Developmental Medical Wisconsin-Madison, Weill and Ophthalmology, of Cell of University of Department Health, Institute, Department Research Public Vision and Dyson Medicine M. Margaret of School Sciences, Visual and Ophthalmology of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. 1 ibryA Toops A. Kimberly , yooe oaiy xctss ebaerpi,A- ltrnaatr NR rti,Fusion protein, SNARE adaptor, clathrin AP-1 repair, Membrane Exocytosis, Polarity, Lysosome, [email protected] 2 ynSchreiner Ryan , 2+ rpnsm cruzi Trypanosoma 1 aei srctst modulate to astrocytes in wave ennoDiaz Fernando , ; [email protected] 2 niu Rodriguez-Boulan Enrique , 2+ ] ´ i idn of binding : uze al., et guez a first was ) 2 oeMraCarvajal-Gonzalez Maria Jose , ) aiekde MC)clsi epnet pore-forming [Ca to (i) in that response demonstrate increase results in Our localized Madin-Darby ionophores. cells calcium polarized and (MDCK) toxins in kidney we exocytosis Here, canine examined. been lysosome yet not membrane investigated has in epithelia wound polarized participate in rapid lysosomes repair necessitating whether lesions, However, toxins, membrane healing. and cause pathogens a with can interact which is directly for survival 2005; that important membranes especially cell epithelia healing is barrier Kirchhausen, for This torn 2005). crucial wound Kirchhausen, and of and response (McNeil conserved in resealing McNeil highly Rapid ubiquitous, 2009; participate 2003). al., also Meldolesi, et may distinct (Divangahi compartments lysosomes other that from show studies repair, membrane 2004). al., 2 et syntaxin (Rao SNAP23, 4 t-SNAREs syntaxin and and VAMP7 v-SNARE the VII, hs tde aeipiain o nesadn lysosome understanding domain. for basolateral the implications to have 4 fusion studies of syntaxin polarity These specifies targeting adaptor accurately clathrin AP-1 by the (iii) syntaxin and of cytoskeleton, localization 4; actin basolateral the the (ii) and on cells; cholesterol predicated membrane MDCK is in exocytosis membrane polarized this basolateral the with exclusively lhuhlssmshv enietfe skyognle in organelles key as identified been have lysosomes Although 2,3, n praLakkaraju Aparna and * 2 ig Gravotta Diego , 2+ ] i nue yooe ofuse to lysosomes induces 1, * 2 , 5937 Journal of Cell Science 5938 ihncdzl lodsutdsnai lses(supplementary clusters 4 syntaxin cells disrupted the also S4). nocodazole not treating with by did microtubules of but depolymerization Fig. the hand, 4, On other S5). D syntaxin Fig. material material (supplementary dispersed distribution cytochalasin LAMP2 and alter filaments that (supplementary actin no confirmed disrupted had exocytosis analysis alone Immunofluorescence drug D on to depolymerizing cytochalasin response actin whereas in effect apically 2A,B), the fuse (Fig. cells, to ionomycin lysosomes MDCK (Low caused membrane In D to basolateral cytochalasin 2006). required the is al., at cytoskeleton clusters et actin intact 4 an syntaxin two, maintain and Muallem 2005; 1995); al., al., et (Ehre et membrane as apical the acts at actin barrier cortical fusion a one, cells mechanisms: two increases non-polarized by exocytosis restricts depolymerization in actin exocytosis (Rodrı and lysosome cytoskeleton calcium-induced actin of remodeling requires the membrane plasma the with fusion Exocytic actin upon lost is depolymerization exocytosis lysosome of Polarity basolateral [Ca exclusive increased 1C; (Fig. to This induced membrane response S2). also in basolateral Fig. (SLO) fusion the material O at supplementary streptolysin exocytosis surface and toxin lysosome stores, calcium pore-forming Thrombin basolateral intracellular cells. the mobilize with MDCK which the predominantly polarized bombesin, in fuse and membrane on lysosomes membrane basolateral that the lysosome-associated exclusively suggesting 1B), of (Fig. LAMP2 appearance protein Ionomycin the cells. untreated to in induced comparable activity was Enzyme medium 1A). apical (Fig. the exposure ionomycin cases, after medium all trans- In ( the (supplementary hexosaminidase S1). monolayer alter Table the not of material does ionophore resistance and electrical cytotoxic S1) calcium not epithelial Fig. is ionomycin the material on concentrations, (supplementary these Cells to At 10 process. media. exposed both or this (5 of were first ionomycin extent we filters and monolayers, Transwell polarity epithelial the been differentiated never established has in exocytosis documented lysosome calcium-induced Because polarized in cells membrane MDCK basolateral the at preferentially occurs exocytosis lysosome Calcium-induced Discussion and Results healing wound pathology, and immunity. and physiology epithelial in function upre ydt hwn that showing data by supported ehnssta ol etitlssm uint the to fusion investigated lysosome then restrict We could lysosomes. surface. preferential basolateral to that of due not S3), mechanisms docking is Fig. LAMP2- exocytosis material basolateral polarized (supplementary that that volume observed suggesting cell uniformly we distributed the were junction, cells throughout MDCK tight in lysosomes the containing the ZO-1 prevent demarcate and at markers that basolateral to mechanisms and machinery apical of Using existence exocytic exocytosis. apical the (Schenkman and/or or asymmetric surface the surface lysosomes basolateral reflect could of basolateral exocytosis distribution Polarized the 1988). al., from et only monolayers ´ uze l,19) nplrzdeihla ci spatially actin epithelia, polarized In 1999). al., et guez ora fCl cec 2 (24) 125 Science Cell of Journal b hx ciiywspeeti h basolateral the in present was activity -hex) m )ete nteaia,bsltrlor basolateral apical, the in either M) rpnzm cruzi Trypanozoma -%o oa cellular total of ,4-6% 2+ ] i yvrossiuiis stimuli various by nesMDCK enters b - otclatni are oeoyoi:atog both although membrane. exocytosis: plasma the fusion, to to accessibility lysosome increasing apical by apical induced presumably barrier the D to cytochalasin whereas 4 only a syntaxin membrane, dispersed membrane, nocodazole is and plasma for D cytochalasin critical actin the are to near microtubules cortical Therefore, response lysosomes colleagues 2002). in and positioning al., Jaiswal exocytose by et demonstrated likely (Jaiswal been membrane has pre-docked as already plasma ionomycin, lysosomes the only cells, near MDCK in exocytosis el u ontpriiaei xctss(aitne l,2011). al., the et and within (Caviston exocytosis locations in material since participate specific long-range Moreover, not to synthesized do lysosomes but for newly cell, as because responsible such transport is organelles This that are 2011). al., exocytosis movements et motors lysosome Laulagnier on 2002; microtubule-based effect al., lysosome et no (Jaiswal of had polarity disruption studies the and with microtubule increased depolymerization agreement alter actin in where cells, are not non-polarized in data did These 2A,B). but (Fig. exocytosis S5), Fig. material tetlsnO(L)o /lthrombin. U/ml 1 or (SLO) 5 O with streptolysin incubation ( of red. minutes in 10 is ZO-1 marker junction Confocal treatment. ionomycin ( cells. and untreated minutes 10 for compartments both (5 Ionomycin cells. MDCK polarized of i.1 yooe uewt h aoaea ebaei epneto response in membrane basolateral the [Ca with increased fuse Lysosomes 1. Fig. 2+ ] B i . perneo AP gen ntecl ufc after surface cell the on (green) LAMP2 of Appearance ) ( A) %o oa cellular total of ,5% b hxatvt naia A)o aoaea B)media (Bl) basolateral or (Ap) apical in activity -hex xy m oprsnof Comparison C) ooyi In) 5 ng/ml 150 (Iono), ionomycin M and b xz m hxatvt esrd o,control Con, measured. activity -hex Mor10 etosaeson h tight The shown. are sections b m hxi eesdupon released is -hex )wsaddt ihror either to added was M) b hxrlaeafter release -hex Journal of Cell Science aaye aeedctctafcb ait fmechanisms of the variety with lysosomes interfere a can which and by Conversely, rabGTPases, of traffic 2012). endosomes inhibition including endocytic al., late et late in Hissa paralyzes 2010; accumulation al., cholesterol et (Chen cholesterol secretion cardiomyocytes, Recent lysosome ionomycin-induced and 2005). increases cyclodextrins fibroblasts al., by depletion in et that (Predescu show cells reports endothelial from agreement in data S6), alteredwith Fig. material (supplementary U18666A apically syntaxin clusters dispersed nor 4 MBCD however, MBCD distribution; actin or Neither LAMP2 lysosome 2A,B). ionomycin-induced (Fig. inhibited exocytosis completely 2008), al., causes (Huynhet that lysosomes and amine endosomes hydrophobic late in a accumulation cholesterol U18666A, contrast, In material (supplementary S4). membrane exocytosis Fig. on effect no of whereas had 2A,B), alone MBCD extraction (Fig. exocytosis lysosome apical cells, caused exposure drives MDCK methyl- and by In cholesterol function traffic. protein organelle modulates in participates biogenesis, cells, mammalian membrane in lipid essential an Cholesterol, membrane levels to cholesterol sensitive is exocytosis Lysosome apical induce extraction cholesterol fusion. and lysosome depolymerization Actin 2. Fig. etblz irtbls yohlsnD(yo)t eoyeieactin depolymerize to methyl- (CytoD) filaments, D cytochalasin microtubules, destabilize aoaea ees rmclstetdwt ooyi alone. ionomycin with treated cells from release basolateral ciiyi pcladbsltrlmda * (B) media. (red). basolateral (Iono) ZO-1 and apical and ionomycin in (green) to activity LAMP2 prior for labeling lysosomes Surface in (A) cholesterol treatment. trap to (U18) U18666A ees rmclstetdwt ooyi alone; ionomycin with treated cells from release ( b A, ccoeti MC)t xrc ebaecoetrlor cholesterol membrane extract to (MBCD) -cyclodextrin B DKclswr rae ihncdzl Nc to (Noc) nocodazole with treated were cells MDCK ) b ccoeti MC)bfr ionomycin before (MBCD) -cyclodextrin P , .1cmae ihapical with compared 0.01 # P , .1 oprdwith compared 0.01, b b -hex -hex rfikn rmteTNadedsmsocr ln a membrane along sort occurs to endosomes cooperate organelles and protein these However, TGN 2007). and 2012; the al., continuum et al., from Gravotta et 2012; trafficking al., (Carvajal-Gonzalez et (CRE) Gravotta endosome recycling hra P1 rmtsei fbsltrlpoen rmthe common the from at proteins proteins epithelia: sorts basolateral AP-1B polarized of (TGN), network exit trans-Golgi in promotes trafficking AP-1A to whereas protein shown basolateral been basolateral have the Taken AP-1B regulates and mediate AP-1A 3D). AP-1 Both (Fig. lysosomes. that of suggest fusion exocytosis data lysosome these together, of polarity Reconstitution basolateral was 3C). (Fig. polarity cells of wild-type of of in secretion secretion basolateral loss non-polar This the m 3B). by (Fig. confirmed either exocytosis of the loss non-polar and that (apical) indicating ZO-1 below, above other one fluorescence, LAMP2 in bumps 7] lhuhLM2lvl in levels LAMP2 Although S7A]. [ cells MDCK of lines stable lacking generated we exocytosis, lysosome or 2009). al., et Laulagnier 1998; Tazeh Carlsson, 2011; and al., (Karlsson of et others in transport not (Ho but lysosomal studies 2002) and some in targeting LAMP1 membrane in participate has has AP-1A 1B identical: 80% are which subunits, yecls lotaltefursec a eo ZO-1 below was fluorescence 3A). (Fig. the cells wild- all in whereas that almost wild-type (basolateral) showed basolateral cells, signal in LAMP2 and type surface localization apical of Quantification the basolateral both its on of LAMP2 membranes of the induced Ionomycin appearance S3). Fig. material (supplementary MDCK ihtefso rcs Fad ta. 00 ern ta. 2002). al., et Lebrand 2010; al., et (Fraldi process fusion the interfere with would which proteins, and SNARE membrane, of plasma sequestration abnormal the at the lysosomes reduce pre-docked thus of and population lysosomes, to motors molecular of recruitment Craa-ozlze l,21;Gaot ta. 02.The 2012). al., AP-1B et epithelial-specific identical the have Gravotta and AP-1A 2012; AP-1A expressed and ubiquitously al., 2007) al., et et (Carvajal-Gonzalez Gravotta 2009; (Fo that Rodriguez-Boulan, AP-1B this shown and by 2008); recently mediated al., basolateral have is et We (Deborde regulation sorted the polarity 1991). then basolateral al., regulates to and et clathrin AP-2 (Nabi transported and lysosomes newly clathrin to first cells, via MDCK endocytosed is polarized membrane, In LAMP2 1998). al., synthesized et (Ohno C- AP-4 (YXXW and via contain ( signals are lysosomes medium LAMPs sorting the to LAMP2 tyrosine-based intermediates. network and terminal trans-Golgi transport LAMP1 the clathrin-coated from proteins transported membrane Lysosomal lysosomes the of regulates exocytosis complex basolateral adaptor clathrin AP-1 The aeilFg 7,) eddntdtc AP ntecell the on LAMP2 of distribution detect intracellular in the not in LAMP2 differences did or 3A) we (Fig. surface S7B,C), Fig. and material wild-type to compared higher m BDand 1BKD BD(rvtae l,20) upeetr aeilFig. material supplementary 2007); al., et (Gravotta 1BKD oivsiaewehrA- atcptsi APtrafficking LAMP in participates AP-1 whether investigate To m Batvt in activity 1B m m B(hoe l,19) Pertinently, 1999). al., et (Ohno 1B A[ 1A m m ADor 1AKD m m aoaea uino yooe 5939 lysosomes of fusion Basolateral c uuiso ltrnaatr P1 P2 AP-3 AP-2, AP-1, clathrin-adaptors of subunits ) AK obekokonclscmae to compared cells knockdown double 1ABKD AD(avjlGnae ta. 02]or 2012)] al., et (Carvajal-Gonzalez 1AKD , m b ADand 1AKD 1, m s BDcls(ize l,20)restored 2009) al., et (Diaz cells 1BKD uuis u ifri hi eim( medium their in differ but subunits, 1 m ADand 1AKD m BDcls oprdt wild-type to compared cells, 1BKD nn ta. 96 uiooe al., et Sugimoto 1996; al., et ¨ning m BDcls ncnrs to contrast in cells, 1BKD m lc ta. 99 Gonzalez 1999; al., et ¨lsch ADcls(supplementary cells 1AKD m m BDclssoe two showed cells 1BKD BDclswere cells 1BKD m uui eutdin resulted subunit 1 b m m hxin -hex Bwssonto shown was 1B A hra AP- whereas 1A, ,wihbind which ), m 1AKD, ,17% m 1B m ) Journal of Cell Science xctssi ltlt Ce ta. 00,ddntco- In not below 4B). (Fig. or at junction did mainly low localizes tight at and 2000), expressed the cells is MDCK wild-type 4 al., in syntaxin levels endogenous et that showed analysis (Chen platelets cargo lysosome with specific immunoprecipitate in implicated a in t-SNARE is a 4 exocytosis 2, syntaxin syntaxin that because show AP-1 with also for data coimmunoprecipitated Our an 4A). 4 (Fig. is 4 syntaxin syntaxin wild-type, whether endogenous In asked we cargo. 2007), AP-1 al., et al., Gravotta et Gravotta 2012; 1999; Because al., 1996). 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(Fig. -adaptin m BDclsepesn myc-tagged expressing cells 1BKD m ADand 1AKD m ADand 1AKD m BDMC cells, MDCK 1BKD m nvivo in ADand 1AKD m BDcls we cells, 1BKD ,syntaxin4 c -adaptin m 1BKD P1.A a endmntae o te aoaea proteins, endosomal basolateral by other membrane for by demonstrated m basolateral TGN been the the has As of to cells, AP-1B. out MDCK sorted transported wild-type and is in AP-1A 4 where, syntaxin model Gravotta synthesized a 2012; newly support al., 2007) et al., Gravotta et 2012; al., et (Carvajal-Gonzalez in exocytosis basolateral and m apical wild-type both in and exocytosis cells, lysosome basolateral inhibited depletion h ci yokltnadcoetrlerce microdomains. cholesterol-enriched and with association cytoskeleton by the actin stabilized At are surface. the clusters apical the 4 al., to syntaxin 4 membrane, et syntaxin of Gravotta mis-localization to 2012; lead the al., overwhelm et could Gravotta In 2012; 2007). al., (Carvajal- proteins et basolateral other Gonzalez to similar the membrane, At apical 2007). in al., and et and Gravotta membrane TGN 2012; the al., et from CRE, (Carvajal-Gonzalez Gravotta 4 CRE 2012; syntaxin the al., et to of trafficking exit increased the promoting mediating by cells) ionomycin shRNA, scrambled in al., secretion the polar et shRNA-mediated with (Torres basolateral treated 4C) used induced (Fig. cells function 4 we In syntaxin syntaxin 2011). secretion, and endogenous of AP-1 lysosome knockdown how polarized elucidate in further To 2011). newy twsrpre htsnai snnoa naporcine were a studies in lacks which nonpolar our LLCPK1, is line, While cell 4 tubule syntaxin protein. proximal that linker reported was a it underway, of participation the or and 4 syntaxin m between interaction direct were a we detect to However, unable S8). 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Scr corresponding ta. 01,poiigfrhrspotfrterl of role the for (Reales support cells further LLC-PK1 providing in 2011), and in al., 1998) apical is al., et 4 syntaxin et Indeed, lack (Fujita 2010). which al., hepatocytes of et both Schreiner 1999; 1998), al., al., et et (Fujita kidney the documented of been has vivo lysosomes in of exocytosis Apical secretion. h oaiyo yooefusion. regulating lysosome and of membrane polarity basolateral the the to 4 syntaxin restricting (actin of 4 absence syntaxin extraction, m of cholesterol redistribution membrane apical depolymerization, cause that Conditions lysosome (WT), for wild-type essential from is immunoprecipitated and AP-1 with interacts exocytosis. 4 Syntaxin 4. 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(Andor 60 (Cytoskeleton, microscopy rhodamine-phalloidin a confocal disk using rat spinning XD) and by wild-type, imaged 1:400) and 4, antibodies Biosciences, syntaxin (BD endogenous 4 of polarity m the determine To staining Immunofluorescence at centrifuged collected, were media basolateral and 100 apical treatments, drug After of Measurement 4 at minutes 30 for of BSA domain medium+1% lumenal recording the in to 1991) antibody al., monoclonal et a (Nabi with LAMP2 incubated were ice on Cells LAMP2 cell-surface of Detection 37 at minutes 10 mM 20 for glucose, (Sigma) g/l 4.5 ionomycin with with (HBSS medium incubated recording and in HEPES) rinsed were Cells ecie lehr Craa-ozlze l,21;Da ta. 09 Gravotta 2009; 2007). al., al., et et Diaz Gravotta 2012; 2012; al., al., et et (Carvajal-Gonzalez elsewhere described of reconstitution lacking lines and cell Stable polarized. otg n fstwr dnia o ie loohr.Zsak eeacquired were Z-stacks fluorophore. given fields, a different for six identical for were 63 offset a were using and XD) voltage Cells Revolution 60 Andor minutes. or or NA 30 SP2 1.4 (Leica for Alexa- microscopy and confocal (Invitrogen) by ZO-1 imaged for antibodies stained secondary and permeabilized Fluor-conjugated paraformaldehyde, 2% with fixed Ca in al., cells to et bound (Idone and exocytosis thiol-activated trigger was (Abcam)] to ng/ml, used [150 was SLO (SLO) 2008). 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Mildred the and (to and Carl Foundation; Foundation; Foundation Kirchgessner Assistance Karl of the to Health E.R.B.); EY08538 (Department American and of the grant UW-Madison) Institutes E.R.B.]; Sciences, core National Visual the P30EY016665 & from Ophthalmology numbers grants [grant by supported Health was work This inspiration. Funding and support invaluable for Lakkaraju Lalita as thank We presented are Acknowledgements data stated, otherwise Unless Prism). mean (GraphPad two-tailed post-test a by analyzed were Data analysis Statistical (Sigma). immunoblotting by antibody confirmed polyclonal was (Origene). rabbit Transwell knockdown a vector to 4 using on pGFP-B-RS Syntaxin plated experiments. a and (Lonza) for into nucleofection filters Amaxa subcloned by were transfected were sequence Cells scrambled a (5 and shRNA 4 Syntaxin interference RNA 300 immunoblotting and Immunoprecipitation rli . nuzaa . obri . asr .J,Mdn,D . Spampanato, L., D. Medina, J., H. Kaiser, A., Lombardi, F., Annunziata, A., Fraldi, W. S. Whiteheart, and T. Schraw, P., P. L. Lemons, E. D., Holzbaur, and Chen, M. Tokito, L., A. Zajac, P., J. A., Caviston, Bay, Perez F., Diaz, R., Mattera, D., Gravotta, M., J. Carvajal-Gonzalez, n rbdfor (30 probed lysates and cell Total for probed SDS-PAGE. and after (Invitrogen) (Sigma) kit immunoprecipitation G Protein Dynabead murcpttdwt 5 with immuprecipitated o lc,H,On,H,Bnfcn,J .adMlmn I. Mellman, and S. J. Bonifacino, H., Davis, Ohno, and C. H., Fo¨lsch, J. Olsen, S., Hill, K., Pestel, De H., L. Abdullah, M., H., A. D. Rossi, C., Lee, Ehre, T., T. Hickman, D., Desjardins, H., Gan, M., Chen, E. M., Falck-Pedersen, Divangahi, J., Schoggins, R., Schreiner, A., Deora, D., and Gravotta, R. F., Schreiner, Diaz, S., Salvarezza, A., Deora, A. D., Y. Gravotta, E., Ioannou, Perret, and S., Deborde, C. Li, W., F. 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Table S1. Trans-epithelial resistance (TER) of polarized MDCK cells cultured on Transwell filters before and immediately after treatment with ionomycin.

MDCK TER (Ω.cm2)

Ionomycin, 10 min Before After

0 µM 114 ± 11.5 113.7 ± 7.8

5 µM 106.7 ± 4.93 109.7 ± 8.74

10 µM 111.7 ± 11.55 106.0 ± 5.29

TER was measured using the EVOM-2 epithelial voltohmmeter (World Precision Instruments). Mean ± SD, n = 3 for each condition.