Journal of Cell Science ebaei amla el Pr n alr 2010). surface Basler, performed cell HEK293 and were and the HEK293T COS-7, (Ba studies (Port as to such mammalian cells non-polarized cells (TGN) these using because network mammalian However, conserved Golgi in transport been their trans membrane supports in have 2010; the and respectively, Wnts Basler, molecules from with (Wls), and interacts Wntless These Wls Port and mammals. 2011). 2011; TMED5 as al., al., evolutionarily, et et Buechling Port 2006; al., et Japan o:10.1242/jcs.126052 doi: 2931–2943 126, Science Cell of Journal 2013 March 18 Accepted n3 nplrzdeihla el srgltdby regulated is mechanisms cells different and epithelial Wnt11 polarized of in secretion Wnt3a basolateral and apical The Article Research n psu Om aebe hw ob eurdfrthe in (ER) for respectively, reticulum required apparatus, endoplasmic be and Golgi the the to from expressed and (Wg) shown Wingless been is of (Evi) have regulates export Wnt interrupted (Opm) and Evenness Opossum how Recently, and pathways contrast, unclear. remains signaling Great In secreted 2007). the functions. by Polakis, mechanisms activates cellular the 2004; understanding Wnt Nusse, (Kikuchi in Wnt which made and humans been in Logan defective have disease advances 2011; and causes al., life processes, et are postnatal that in developmental molecules signaling secreted of in family large important the are proteins Wnt Introduction that and Wnt3a and Wnt11 of secretion polarized of the the for site not in glycosylation but words: whereas roles Asn40 Wnt3a routes. Key different Wnt11, of the secretory has secretion of of their high- basolateral Wls fusion decides the secretion that for the Wnts apical complex/hybrid(Asn40)-, suggest necessary By of was results the Wnt3a. processing the AP-2 These with by for of glycosylation Wnt11. to Wls secretion required of of basolateral modified localized recycling secretion were The the glycans apical was galectin-3 apically. for is high-mannose-type secreted and Wls required was two Wnt3a were Asn40 cells. Wnt11 with Wnt11, protein-1 at modified epithelial adaptor processing that is and polarized mechanisms. Glycosylation Wnt3a clathrin different in Asn298). revealed that by respectively, regulated and and is of analyses glycans (Asn87 basolaterally, be Wnts secretion high-mannose/hybrid(Asn300)-type to of polarized and shown secretion Mass-spectrometric and the apically was basolateral in mannose(Asn90)- Glycosylation and roles secreted membrane. clarified. apical their of the been and were polarized role show not profiles basolateral Wnt3a the we in glycan have Here secretion, but cells secreted and demonstrated. proteins, their epithelial are been secreted support Wnt11 polarized and yet Wnts and in transmembrane not Wnts destination how some has with final but of Wnts interact the sorting responses, to to the shown cellular Wnts for been of for important has sorting important (Wls) the are Wntless in Although that Wls unclear. morphogens remains lipid-modified cells and epithelial glycan- are Wnts Summary Shim Mibu, Kitakobayashi, 880 University, Medical Dokkyo Medicine, of School Graduate Biology, Cell ` and Molecular Japan of Department address: *Present 2 1 Yamamoto Hideki ohfm Takao Toshifumi uhrfrcrepnec ( correspondence for Author aoaoyo rti rfln n ucinlPoemc,IsiuefrPoenRsac,OaaUiest,Ymdoa32 ut,Oaa565-087 Osaka Suita, 3-2, Yamadaoka University, Japan Osaka 565-0871, Research, Osaka Protein Suita, Yamadaoka, for Institute 2-2 Proteomics, University, Osaka Functional Medicine, and of Profiling School Protein Graduate of Biochemistry, Laboratory and Biology Molecular of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. nie ta. 06 omse l,21;Jne l,21) the 2010), al., et Jin 2010; al., et Coombs 2006; al., et ¨nziger lcslto,Plrt,Scein n,Wntless Wnt, Secretion, Polarity, Glycosylation, 2 [email protected] 1 n kr Kikuchi Akira and hhr Awada Chihiro , Drosophila 2 iek Hanaki Hideaki , 1, ` ) (Ba ¨nziger 1 ioh Sakane Hiroshi , ebaepoen He ta. 03 ai ta. 2009). the cells in epithelial al., implicated in proteins been membrane et apical have glycosylated 1999) of Vagin sorting Hughes, 2011; 2003; al., et recognize al., (Boscher that galactic-4 et and Galectin-3 (Huet proteins membrane as in identified such signals, been basolateral have instance, signals Yxx For sorting proteins. Several basolateral membrane 2005). and Rodriguez-Boulan al., apical 2012; al., of et et incorporation (Cao in vesicles by distinct Wnts TGN into proteins the of of level secretion the basolateral understood. well and not apical are cells in epithelial polarized Wls of roles ta. 02.Aia inl,sc sglycosylphosphatidylinositol as such 2008; anchors, signals, Apical al., 2002). et al., et membrane Deborde different 2001; of Bonifacino, to Fo signals and proteins basolateral (Boehm direct with domains and interact (APs), proteins AP-4, membrane complexes and clathrin- protein AP-1 and of including adaptor Clathrin region heterotetrameric cytoplasmic proteins. the associated membrane in present plasma are basolateral motifs, LL and chains) lc,20;Gaot ta. 02 rvtae l,20;Simmen 2007; al., et Gravotta 2012; al., et Gravotta 2005; ¨lsch, h otn rcs noteaia n aa ein cusat occurs regions basal and apical the into process sorting The W ( W N gyasand -glycans ersnigayaioai ihhdohbcside hydrophobic with acid amino any representing 1, ,IuoTsujimoto Ikuko *, O gyas aebe eotdi apical in reported been have -glycans, 1 uoTakahashi Yuko , b glcoiestructures -galactoside -sg,Tcii321-0293, Tochigi o-tsuga, 2931 1 1, , Journal of Cell Science sesnilfrtesceino n1 n n3,Wsis not Wls Therefore, but TGN. Wnt3a, the membranes from and basolateral exit Wnt11 their the after of Wnt11 to with polarized secretion associated Wnt3a 1F; Wls (Fig. in the with although that was for trafficked mainly suggest Wnt3a essential results Wls membrane These is and 1G). basolateral Interestingly, (Fig. cells Wnt11 the S1A). MDCK in of Fig. S1A). observed material secretion Fig. rescued transferrin cells supplementary material polarized MDCK and Wls-depleted supplementary in podocalyxin the Wls human 1F; of of Fig. polarized Expression not sorting (TfR; in did knockdown polarized receptor basolaterally the which the Wnt3a in affect conditions and under cells of apically MDCK secretion the Wnt11 inhibited completely both almost Wls of Knockdown apical (Ba cells an non-polarized is that of ezrin secretion polarized Wnt3a. of the and support localization Wnt11 results 1D), These the (Fig. 1E). to (Fig. kidney of marker 1C). similar embryonic region (Fig. mouse apical is membranes, membranes the of which in basal epithelium basal located bud the be ureteric to but on and tended detected Wnt3a, Wnt11 apical Endogenous was permeabilization, Wnt11, were Without of not cells 1C). vicinity (Fig. in the detected respectively When were immediate Wnt3a 2002). and Wnt11 the al., stained, be et to permeabilized (O’Brien 1B). cells. little Matrigel (Fig. very any MDCK using if but fraction, fraction, in basolateral apical the Wnt3a in the detected in suggesting and was observed apical Wnt11 1B), Wnt11 also the (Fig. was in of Wnt3a The respectively detected secretion was 1B). fractions, (Fig. Wnt3a polarized basolateral and agarose Wnt11 and NeutrAvidin the of using biotinylated majority were the precipitated membranes basolateral in and and apical media were the membrane-associated surface culture on Wnts cell Wnts and soluble from chambers, lower Secreted Sepharose and upper 1A). Blue (Fig. support using 1995) filter a precipitated al., on monolayer cells, et a as epithelial (Gottardi cultured were polarized expressing Wnt3a cells or in epithelial Wnt11 either (MDCK) fraction into kidney basolateral canine secreted Madin–Darby are or Wnt3a apical and the Wnt11 whether Wnt3a examine and To Wnt11 of secretion Polarized secreted are we Results Wnt3a and Here mechanisms. mass different Wnt11 by by respectively, cells. Wnt3a that basolaterally, and and apically show epithelial Wnt11 or 2002). and of apical polarized the profiles spectrometry Lin, to glycan secreted in the and 2001; are demonstrate al., Wnts region Vainio how et clear 2003). Lickert basolateral 2012; not 2011; is cells al., al., Kypta, it epithelial However, et in and et Hayashi present 2005; Uysal-Onganer are Veeman al., Wnt11 et and 2009; (Dean Wnt3a al., that al., et revealed and Kikuchi et 2000; Immunohistochemical MacDonald al., et 2011; (Heisenberg pathways canonical) apical the be to suggested 2003). were al., proteins et (Huet secreted sorting signals unclear. some proteins, remain membrane in proteins to glycans secreted contrast In for 2009). signals 2005; al., al., et Delacour et 2007; al., Stechly et Delacour 2006; al., et (Delacour 2932 l neat ihWt n sesnilfrtersceinin secretion their for essential is and Wnts with interacts Wls culture (3D) three-dimensional in cyst a formed cells MDCK activate that ligands representative are 11 Wnt and Wnt3a b ctnndpnet(aoia)ad-needn (non- -independent and (canonical) -catenin-dependent ora fCl cec 2 (13) 126 Science Cell of Journal nie ta. 06 otadBse,2010). Basler, and Port 2006; al., et ¨nziger nsitu in yrdzto analyses hybridization N gyasand -glycans O - rae ihPGs rEd H were Endo cells or MDCK of F CM PNGase the with into secreted treated Wnt3a and Wnt11 l a aedsic oe nteplrzdsceino Wnt11 of secretion polarized the in Wnt3a. roles and distinct have may Wls oiiysita nsfo Mo el (supplementary cells L of CM from Wnts as shift mobility aeilTbe 2 3.Bsdo eiso asvle ihthe with values mass of series (supplementary a on procedure Based Wnt3a, and S3). this and S2, 82.1% Wnt11 Tables by material of about recovered sequences acid were covered amino respectively, which entire the peptides, of 76.9% tryptic A laser of (MS)/MS. reversed-phase matrix-assisted resulting number spectrometry by nano-flow mass the (MALDI) followed 2007). to and desorption/ionization (LC) al., subjected trypsin, chromatography et were with liquid Komekado peptides digested 2004; al., were tryptic from et Wnts purified Table (Kishida Purified S2A–C; Wnt3a were Fig. stably or cells) material Wnt3a S1) (L (supplementary fibroblasts and Wnt11 mouse expressing of Wnt11 and possible (CM) N87 medium Wnt3a), the and conditioned Wnt11, Asn-linked at for for N304 profiles and N298 N300 for glycan N160, N90, the (N40, sites respectively, analyze To consensus potential glycosylation. (Asn-X-Ser/Thr), two and five are suggested sequences there been and have Wnt3a glycosylated and be Wnt11 to al., 2009). et al., (Huet et proteins Vagin secreted 2003; and transmembrane some of sorting Wnt3a and Wnt11 N of lipidation and Glycosylation lae -ikdgyas n nolcsds H endoglycosidase and glycans, by N-linked gel Peptide- cleaves SDS-polyacrylamide with the treatment on the patterns migration (supplementary different Wnts other is in Wnt11 conserved are in S3). not Wnt3a Fig. N40 is in material whereas it N298 Wnts, that and in mouse N87 unique 19 and S4, in Wnt11 Tables conserved in highly N300 material and be be (supplementary N90 to to Wnt3a S5). types of Wnt11 N298 high-mannose and of N87 both to N300 attached and those types, and high-mannose/hybrid N90 respectively, and N40, glycan high-mannose to The complex/hybrid, attached 2D,E). indicated values (Fig. Wnt11 glycans were mass Wnt3a for observed the intervals for the (Sia) from whose Hex acid estimated with compositions peaks, sialic and and ion 2A–C) a HexNAc (Fig. of molecular Hex, Wnt3a N300 of of of with N298 and consistent series and N90 glycans N87 a al., with N40, glycopeptides et gave the two with (Satomi the glycopeptides and masses of Wnt11 three molecular The abundances their the 2004). on for relative allows based method glycopeptide of ESI nano- to the subjected estimation because were in digests LC/ESI-MS/MS, tryptic observed the flow Wnt3a, LC/ and not The Wnt11 nano-flow of glycosylated. using was not observed Wnt3a were (ESI)-MS/MS. was ionization Wnt11 of but electrospray turned of measurements N87 Wnt3a N300 these of N304, containing of N298 N160, part peptide whereas and glycosylated, a Wnt11 heterogeneously and of be N300 to out and of N90 masses residual N40, the represent and which (Hex) 203, hexose and 162 of intervals xiie h aemblt hf hni a rae with treated was it not when Wnt3a H shift but S4A). Endo mobility or types Fig. F same PNGase material hybrid the (supplementary and exhibited type high-mannose complex the the cleaves which Gyoyainwsrpre ob novdi h polarized the in involved be to reported was -Glycosylation ossetwt h eut fM nlss n1 showed Wnt11 analyses, MS of results the with Consistent site glycosylation each at profiles glycan the clarify further To N aey eoaie(eNc,respectively, (HexNAc), hexosamine -acetyl f splmnaymtra i.SA.When S4A). Fig. material (supplementary N gyoiaeF(NaeF,which F), (PNGase F -glycosidase f hysoe h same the showed they , f Ed H (Endo f ), Journal of Cell Science eesanduigat-zi gen n anti- and (green) anti-ezrin Wnt3a. using and Wnt11 stained of were secretion Polarized 1. Fig. ( HP0 a sda odn oto.( control. loading a as used signa was the or (HSP90) fraction apical control the in Sepharose were Wnts membrane-associated and Sepharose, Blue aoaea ebae a e to set was membranes basolateral subj were cells the siRNA, Wls or control with transfected were Wls–MycHis tr and pr Podocalyxin N, 100%. Sepharose; to Blue set using were precipitation fractions basolateral and e apical were the results into the and ImageJ NIH using quantified fmuekde tE3wt niWt1(re)a (green) anti-Wnt11 with E13 expr at indicate kidney Arrowheads mouse right. of the on image wer the 13 in (E) magnification day embryonic at kidney mouse of st sections were treatment permeabilization without or B DKclsepesn n1 rWtawr subjec were Wnt3a or Wnt11 expressing cells MDCK ) 0% h eut hw r means are shown results The 100%. cptto sn etAii grs.( agarose. 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Journal of Cell Science htWt1i oiidwt amtli cda Ser215. at acid palmitoleic with modified indicating was is 2G), lipidation Wnt11 (Fig. MALDI-MS/MS of that by site S215 The at products. assigned oxidized clearly air the of observation novmn fgyoyainadlpdto fWt1and Wnt11 of lipidation and glycosylation of Involvement the at be to whose acid, determined palmitoleic was (C16:1), bond acid double fatty mono-unsaturated erto,gyoyainstso n1 eemttdt l (Q) for Gln ER to the mutated in were Wnt11 Wnt11 of of sites glycosylation glycosylation of secretion, role the investigate trafficking To intracellular the in Wnt3a D oiinby position 9 uain lce t erto Fg C Kmkd tal., double et WT but expressing (Komekado cells secretion, L 3C) either affect of (Fig. of supernatant not post-nuclear secretion mutation When did A its 2007). was Wnt3a 3B). blocked (Fig. in Wnt11N300Q/N304Q mutations Wnt11 N298 3B). 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(Fig. not did inhibitors xrsigWt1wt iuesn rdM eutdi the in H Endo resulted cells to dMM, sensitive MDCK or becoming the of kifunensine Wnt11 of secreted with treatment accumulation Wnt11 the The expressing in glycans. Fuhrmann results and high-mannose-type 1990; and hybrid- of al., with glycans synthesis the et prevent complex-type treated inhibitors Elbein These 1984). 1984; al., were et Kornfeld, inhibit cells and which (Bischoff profiles, (dMM), glycan deoxymannojirimycin ER Wnt3a and and the Wnt11 kifunensine of on secretion polarized depends whether examine Wnt11 To for of N40 secretion at apical glycan the complex/hybrid-type the Wls. of Requirement with interaction functional Wls its for with 3D). necessary complex in (Fig. is acid a palmitoleic Wnt11 fractions Therefore, formed S5B). ER Wnt11S215A Fig. material the Wnt11S215A not (supplementary and but and Golgi Wnt11 S5A) the WT in Fig. in secretion accumulated material its was suppressed (supplementary Wnt11 cells in S215 al., L et of (Coombs mutation Wls A with interaction 2010). and secretion its exit. for required ER the (N87 for Wnt3a and necessary and ER, also N300) the and is (N90 in N298) of Wnt11 N40 and exit in to glycans ER two glycan the of of Therefore, either attachment was 3D). the (Fig. Wnt3aN87Q/N298Q, requires fraction as Wnt11 3D). ER well (Fig. the fractions as in ER Wnt11N40Q, detected and the Golgi of cytosol, was the Most Wnt3a of WT or all was Wnt11 in WT Wnt3aN87Q/N298Q observed gradient, or sucrose a Wnt11N40Q on fractionated Wnt3a, WT Wnt11, 2936 oeaiewehrteadto ftegyoyainsite glycosylation the of addition the whether examine To n-,wihi n fsix of one is which Gnt-I, h oiiainwt amtli cda 29o n3 is Wnt3a of S209 at acid palmitoleic with modification The a mnoiaeIadGolgi and I -mannosidase ora fCl cec 2 (13) 126 Science Cell of Journal ˚ oboktesceino rtisa the at proteins of secretion the block to C N aeyguoaietaseae in transferases -acetylglucosamine a mnoiaeI respectively I, -mannosidase ˚ ,adtetetof treatment and C, f hra these whereas , A eimsbnt fA-AadA-B u not but AP-1B, and AP-1A 2002). of al., subunits et medium Simmen HA, 2007; al., Fo et 2008; Gravotta al., the Bonifacino, et to and Deborde proteins (Boehm 2001; cells cargo epithelial sort in AP-4 membrane and basolateral AP-1B Wnt11 of AP-1A, secretion and apical the the Clathrin for for not Wls but and Wnt3a of AP-2 secretion AP-1, clathrin, of Requirement o o hto n1,fo h G.Cnitn ihthese in decreased with not Consistent was TGN. 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(supplementary slightly Fig. clathrin material of with (supplementary interacted clathrin Wls–MycHis endogenous and ,ide omdacmlxwt l–yHsadendogenous adaptin- and Wls–MycHis and with complex clathrin a formed 2008). indeed al., 2, et Yang 2008; al., in et secretion Wg for required and is and Wls TGN AP-1. of with the endocytosis and 6C; mainly from clathrin (Fig. Wls, secretion membrane of Wnt3a TfR complex basolateral the that the by and and regulated to AP-1 conceivable sorted and Wls is clathrin is it 6A,B). 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Fig. aebe lcsltdisedwe 30wsmutated. was N300 might the when N304 instead because is in results, glycosylated correct been Wnt the residue have provide purified always Asn not until glycosylation may for the sites analyses glycosylated Mutational that spectrometry. indeed mass by concluded analyzed is be sequence cannot consensus not it was Therefore, glycosylation their Wnts. sites, other from different the be Therefore, might Wnts. Wnt11 other was of not of structure Wnt11/3a does structure folded region the Because N-terminal affect the to Wnt11. to important appear glycan of be of of addition could integrity exit the site secreted, ER structural this the to the for glycan essential for of was attachment ER Because the the region. 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Wls and al., of et Port recycling the Yang 2008; 2008; al., al., et et Port (Pan 2010; retromer sort the clathrin/AP-2-mediated could cell with by the Golgi endocytosis clathrin from the and up and endosomes taken the also AP-1 to is surface Wls Wls, membrane. plasma of the to complex is Wnt3a tertiary Wls AP-1, Therefore, of and the clathrin Wnt11. with and secretion mainly of membrane basolateral secretion basolateral the apical to sorted and the not apical but Wnt3a the suppressed molecules thsbe eotdthat reported been has It of transport the promotes Wls that demonstrated also was It ora fCl cec 2 (13) 126 Science Cell of Journal m id otetrsn-ae otn inlof signal sorting tyrosine-based the to binds 1 Drosophila m n ltrn n ncdw fthese of knockdown and clathrin, and 1 N iceihlu Gle ta. 2008). al., et (Gallet epithelium disc gyasaeaia otn inl in signals sorting apical are -glycans lc,20) l formed Wls 2005). ¨lsch, b neat with interacts 1 neoye Dlcu ta. 08 idtd ta. 1993). cells. epithelial al., in of roles polarity et plays apicobasal Wnt11 the Lindstedt that of speculate maintenance 2008; to the intriguing al., is it of et Therefore, membrane galectin-3 lateral (Delacour the and of into villin perturbation enterocytes intestine, marker and border LPH mouse brush apical of the of mislocalization the border causes physiological knockout brush detected the is the is Galectin-3 Wnt11? the What of beneath secretion TGN. inhibiting apical the the of by Wnt11-loaded from relevance when released membrane complex are Wls basolateral the vesicles with the Wnt11 from of to Wnt11 interaction prevent sorted might N40 being at The mechanisms. glycan are different by Wnt3a complex/hybrid-type destinations and final Wnt11 their Thus, to AP-2. trafficked and the AP-1 clathrin, in require not involved membrane. apical be the might to Wnt11 galectin-3 galectin-3- of with transport and fused vesicles, vesicles endosomal containing the in galectin-3 with nvriy e ae,C,UA n .Tkd Ntoa ntttso Natural of Institutes (National pCB6/ Takada and pCB6/ S. pEGFPc1/galectin-3, respectively. and Japan), Okazaki, USA) Sciences, CT, Haven, (Yale Wu New D. University, Drs by provided were Wnt3a pPGK-neo/mouse and Wnt11 pCS2/mouse chemicals and Materials methods and Materials otasetsRAit DKclsepesn n1,Wta n1/aor Wnt11/3a Wnt3a, at Wnt11, (Invitrogen) Optimem expressing in suspended cells were cells MDCK MDCK trypsinized into Wls–MycHis, siRNA siRNA by transfect cells MDCK To in proteins of Knockdown cells the 800 containing Then, medium lentiviruses. same the with in maintained infection and by selected Wnt3a were generated co- WT stably co-expressing were and cells Wls–MycHis mutants GFP–galectin-3 MDCK or their with G418. Gnt-I or with with Wnt3a Wnt11 selection WT WT by Wnt11, expressing 10% (FBS). generated WT with were expressing serum supplemented Wls–MycHis stably bovine cells and F12 modified MDCK DMEM/Ham’s fetal or Dulbecco’s in L 10% in FBS. maintained with maintained were cells supplemented were HEK293T cells (DMEM) NIH3T3 medium and Eagle’s X293T L, MDCK, culture Cell H Endo and F PNGase (San Cruz respectively. Santa and USA), from USA) CA, MA, were (Carlsbad, antibodies Diego, Invitrogen anti-podocalyxin UK), and Anti- (Cambridge, USA). (TfR) Abcam anti- MA, receptor (Beverly, and Technology anti-transferrin anti-Wnt5a/b Signaling immunoblotting), ezrin, Cell from (for were Anti-Wnt3a antibodies USA). Dvl2 respectively. CA, Covance USA), Jose, for (San MO, from Louis, (16B12; purchased (St. adaptin- Anti-HA Sigma-Aldrich were respectively. and Anti- antibodies cells, USA) NJ, 12CA5 anti-FLAG were (Princeton, and antibodies and by immunoprecipitation) 9E10 rabbits immunoblotting) (for in from anti-HA generated and generated mouse 2004; was (tag) of 141–157 antibody al., Anti-Myc residues to anti-Wnt11 et Wnt11. corresponding peptide An synthetic (Kishida a 2007). with previously immunization al., described et as Komekado generated was immunostaining) respectively. Japan), Nagoya, Brook, University, Stony (Nagoya York, Kaibuchi New K. of University and (State USA) Hsieh C. NY, (Advanced J. pGEX- Taniguchi Drs and by provided pRK5/IgG N. were Japan). CRIB and Tsukuba, Technology, Narimatsu and H. Science Industrial Drs from Fo was pcDNA3.1hygro/Gnt-I H. and 2.5 hssuyaesoni upeetr aeilTbe 6 7 epciey Other respectively. S7, sources. S6, in commercial Tables from used material obtained RT-PCR supplementary were quantitative in materials to for shown primers previously used are reverse The study were and mutants. this described forward techniques its and or DNA duplexes, Wnt11 as RNA recombinant pPGK-neo/WT Standard and pPGK-neo/Wnt3aS209A constructed pCGN-HA 2007). construct into al., subcloned was et and (Komekado (Invitrogen), to pcDNA3.1/hWls-MycHis library pCSII-CMV-MCS-IRES2-Bsd cDNA Human human vector. into Japan). a Shiga, from cloned Inc., Bio cloned (Takara were vectors lentiviral cDNAs were construct GFP-galectin-3 Gnt-I, agarose Wls-MycHis USA). 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Zanetta, V., Gouyer, D., Delacour, ucln,T,Caday . prh,K,Wis .adBurs M. Boutros, and M. Weiss, K., Spirohn, V., Chaudhary, T., Buechling, eod,S,Pre,E,Gaot,D,Doa . avrza . crie,R and R. Schreiner, S., Salvarezza, A., Niswander, Deora, D., and Gravotta, A. E., R. Perret, Lang, S., D., Deborde, Dufort, N., A. Smith, A., L. Miller, H., C. Dean, oms .S,Y,J,Cnig .A,Vlr,C . oe,T . hog .K., J. Cheong, M., T. Covey, A., C. Veltri, A., C. Canning, J., Yu, S., K. G. Simons, Coombs, and A. M. Surma, X., Cao, R. I. Nabi, and W. J. Dennis, C., Boscher, S. J. Bonifacino, and M. Boehm, D., Yan, R. V., Kornfeld, Y. and J. Sharma, Bischoff, L., Cheng, B., Zhou, X., Tang, Y., Wu, Y., T. Belenkaya, T. Ba Takao, and T. Sato, C., Awada, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126052/-/DC1 online available material Supplementary to Scientific 23112004 Research for number [grant and Scientific (2011-2012) A.K.]. A.K.]; Areas for to Priority on 21249017 Grants-in-Aid Research number through and [grant Science, Japan (2009-2011) Education, of of Ministry the Culture by supported was work This Funding manuscript. provided the and wrote mass-spectrometry and and experiments by designed assay Wnts A.K. advice. of sorting profiles glycosylation Wnt lipidation analyzed apical-basolateral T.T. and of and C.A. purification out assay. fractionation performed and proteins carried Y.T. assay. I.T. immunohisotochemical H.H., and manuscript. proteins the wrote Wnt H.S. and of assay, purification sorting apical-basolateral out and for carried experiments, Kaibuchi designed Fo H.Y. K. H. and contributions Fenteany, Author Hsieh G. C. Takada, J. S. Taniguchi, plasmids. Wu, N. donating D. and Drs Narimatsu thank We Acknowledgements 2010). al., et density (Matsumoto between complex previously Sucrose a described of 2007). Formation except used. described 1A-HA, was al., 2007) (10–40%) Wls-MycHis, al., as gradient et et sucrose (Komekado performed linear Kurayoshi described a that as were 2007; (ECM) performed was cells matrix al., fractionation were extracellular gradient Wnt-producing et X-114 the (Komekado from kidney Triton CM, previously The lysates of embryonic 2009). preparation and al., mouse and fraction, et assay in (Yamamoto separation previously phase Wnt11 described as of performed studies Immunocytochemical statistical methods for Other tested were data the significant. statistically between using Differences significance Inc.). Institute (SAS 2942 nie,C,Slii . Schu D., Soldini, C., ¨nziger, n uftdsi pclmmrn rfikn netrct-iecells. enterocyte-like in trafficking membrane apical 169 in sulfatides and oeuHneoce . as . os . Andre A., Pons, E., Maes, O., Moreau-Hannedouche, Nature E. Rodriguez-Boulan, gland. lacrimal and lung the of A. L. tm,V,Bnre,N,Zag .H,Jdlo .C tal. et C. R. 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