Supplementary Figure Legends

Transcription

1 Supplementary Figure Legends Supplementary Fig. 1. The third PDZ domain of PAR-3 and the C-terminal PDZ domain binding motif of VE-cadherin mediate the recruitment of PAR-3 to cell-cell contacts in cells. (A) The third PDZ domain of PAR-3 is necessary for its localization at VE-cadherin-based cell-cell contact sites. HeLa cells were transiently transfected with JAM-A (top panels) or VE-cadherin (bottom panels) expression vectors together with recombinant Myc-tagged PAR-3 constructs containing either wt PDZ domains 1 to 3 (PAR-3/wt) or mutant PDZ domains 1 (PAR-3/mt1), 2 (PAR-3/mt2), or 3 (PAR- 3/mt3). Ectopically expressed proteins were visualized by indirect immunofluorescence using antibodies against JAM-A, VE-cadherin or the Myc epitope for PAR-3 constructs. Bars, 10 µm. (B) HeLa cells were transiently transfected with Myc-PAR-3/PDZ1-3 and either full length VE-cadherin (VE-cad/wt) or VE-cadherin lacking C-terminal 84 AA including the β-catenin binding site and the PDZ domain motif (VE-cad/ 84) or VE-cadherin lacking C-terminal 5 AA comprising the PDZ domain binding motif (VE-cad/ 5). As controls, the VE-cadherin constructs were co-transfected with β-catenin (middle panel) or p120 ctn (bottom panel) as indicated. Ectopically expressed proteins were visualized by indirect immunofluorescence using antibodies against VE-cadherin, β-catenin, p120 ctn or the Myc epitope for PAR-3 constructs. PAR-3 recruitment by VE-cadherin is abolished upon deletion of the PDZ domain-binding motif whereas β-catenin and p120 ctn are still recruited. Bars, 10 µm. Supplementary Fig. 2. Characterization of the PAR-6 binding site in VE-cadherin. (A) Both PAR-6α and PAR-6γ directly associate with VE-cadherin. GST-pulldown experiments were performed with GST-fusion proteins and in vitro translated, 35 S-methionine-labelled PAR-6α, PAR-6γ or PAR- 3/PDZ1-3. Both PAR-6α and PAR-6γ associate directly with VE-cadherin. (B) PAR-6 binds to the membrane-proximal and part of the middle region of the cytoplasmic tail of VE-cadherin. GST-fusion proteins encompassing various regions of the cytoplasmic tail of VE-cadherin (pm, AA , pm1, AA , pm2, AA , pm3, AA , pm4, AA ) were incubated with in vitrotranslated PAR-6α (top panel) or p120 ctn (bottom panel). PAR-6α associates with VE-cadherin through AA of VE-cadherin. The binding of p120 ctn to VE-cadherin constructs pm, pm1, pm2 and pm3 but not pm4 is consistent with the presence of the complete p120 ctn core binding region in pm, pm1, pm2 and pm3 but not in pm4. (C) Schematic summary of the VE-cadherin PAR-6 interaction (experiments depicted in Fig. 3C and Suppl. Fig. 2B). Supplementary Fig. 3. The distribution of ICAM-2 as apical marker does not change upon prolonged culture of EC. To analyze the distribution of the apical membrane protein ICAM-2, bend.3 EC were cultured for different time periods and analyzed for ICAM-2 localization by indirect

2 immunofluorescence microscopy. Cells were seeded at a density of 5 x 10 4 cells per cm 2 on 0.45 µm polycarbonate filters and cultured for two days (top panel) or seven days (bottom panel). Cells were fixed and stained with antibodies against ZO-1 and ICAM-2 as indicated and analyzed by confocal microscopy in x-y-sections (squares) and x-z-sections (rectangles). The apical localization of ICAM-2 does not change during this culture period indicating that the formation of apico-basal polarity does not change during prolonged in vitro culture of EC. Bars, 10 µm.

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6 Supplementary Information Antibodies and plasmid constructs The following antibodies were used: rabbit pab anti-par-3 (Upstate), rabbit pab anti-par-3 C2 and anti-par-3 P2, rabbit pab anti PAR-6B/β {Yamanaka, 2003 #524}, rabbit pab anti apkcζ C-20, anti-par-6 T-20 (all from SantaCruz), rabbit anti ZO-1 (Zymed), mouse mab anti-apkcλ, anti-αcatenin, anti-β-catenin, anti-p120 ctn, anti-calretinin, anti-calreticulin, anti-human CD144, anti ICAM- 2 (BD Biosciences), anti-golgi 58K protein (Sigma), anti occludin (Zymed), rabbit pab anti-phosphoapkcζ/λ (Cell Signaling Technology), rat anti-e-cadherin (DECMA-1, Sigma), anti PECAM-1 (Mec13.3, Pharmingen), rat anti VE-cadherin 11D4.1 {Baumeister, 2005 #704}, mouse anti myc epitope 9E10 (BD PharMingen), rat mab anti JAM-A {Ebnet, 2001 #530}. Rabbit polyclonal antibodies against PAR-3 and PAR-6 were generated by immunizing rabbits with GST-PAR-3 (AA ) or GST-PAR-6C/α fusion proteins, respectively. GST-fusion constructs encoding GST-JAM-A, -JAM-B, -JAM-C, VE-cadherin, N-cadherin have been described elsewhere {Ebnet, 2003 #505; Baumeister, 2005 #704}. The following GST-fusion constructs were generated in pgex-4t-1 or pgex-6p-2: VE-cad/ 20 (AA ), VE-cad/ 5 (AA ), VE-cad/pm (AA ), VE-cad/md (AA ), VE-cad/p (AA ), VE-cad/m (AA ), VE-cad/d (AA ), E-cad (AA ), E-cad/VE-cad-5 (AA of E- cadherin followed by AA of VE-cadherin), VE-cad/C10 (AA ), E-cad/C10 (AA ), and Pals-1 (AA 1-182). Eukaryotic expression constructs encoding full length VE-cadherin, E- cadherin, β-catenin, α-catenin, p120 ctn and PECAM-1 were cloned into pcdna3. Expression vectors encoding PAR-3 full length, individual PAR-3 PDZ domains and PAR-3 PDZ domain mutants have been described previously {Ebnet, 2001 #530}.