Figure S2.
Quantification of Src—JAM-A and Csk—CD9 association as well as of Src, Erk1/2 and Abi1 phosphorylation in JAM-A-depleted cells. (A) Quantification of Src association with JAM-A in the absence and presence of VEGF stimulation (addendum to Fig. 4 C). (B) Quantification of Src phosphorylation (Tyr530) in JAM-A KD MCF7 cells (addendum to Fig. 4 F). Note that Tyr530 of Src is a negative regulatory Tyr residue, and that low pY530/total Src ratios are indicative for high Src activity. (C) Quantification of Erk1/2 phosphorylation (Thr202/Tyr204) in JAM-A KD MCF7 cells (addendum to Fig. 4 G). (D) Quantification of Abi1 phosphorylation (Ser225) in JAM-A KD MCF7 cells (addendum to Fig. 4 H). (E) Quantification of Erk1/2 phosphorylation (Thr202/Tyr204) in JAM-A KD MCF7 cells treated with PP2. (F) Quantification of Abi1 phosphorylation (Ser225) in JAM-A KD MCF7 cells treated with either CI-1040 or with PP2. (G) Quantification of Csk association with CD9 in the absence and presence of VEGF stimulation (addendum to Fig. 5 A). (H) Paxillin localization in MCF7 cells cultured on VN-coated micropatterned substrates (5 µm width). Arrowheads indicate polarized enrichment of paxillin at the periphery of cells migrating on the spatially confined substrate. Scale bars: 10 µm. (I) Paxillin phosphorylation at Tyr118 and Ser126 after inhibition of ERK1/2 (CI-1040) or Src (PP2). Top: Western blot analysis using antibodies against total paxillin (Pax), P-Ser126 paxillin (pS126Pax), or P-Tyr118 paxillin (pY118Pax). Bottom: Quantification of paxillin phosphorylations. Western blot signals were quantified using the Odyssey application software. In A and G, signals obtained with Src and Csk antibodies were normalized to the signals obtained with JAM-A and CD9 antibodies, respectively. In B, C, D, E, F, and I, signals obtained with phospho-specific antibodies were normalized to the signals obtained with antibodies directed against the respective total protein in each experiment. (J) Cell size analysis of MCF7 cells stably transfected with a control vector (pLVTHM-EGFP, Control KD) or with a JAM-A shRNA expression vector (pLVTHM-EGFP-JAM-A-shRNA, JAM-A KD) seeded on VN-coated or FN-coated polyacrylamide gels (for VN: n = 34 for Ctrl cells, n = 35 for JAM-A KD cells, four independent experiments; for FN: n = 74 for Ctrl cells, n = 74 for JAM-A KD cells, four independent experiments). Data is taken from three independent experiments in each panel. Statistical analysis was performed using unpaired Student’s t test. NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Source data are available for this figure: SourceData FS2.

Quantification of Src—JAM-A and Csk—CD9 association as well as of Src, Erk1/2 and Abi1 phosphorylation in JAM-A-depleted cells. (A) Quantification of Src association with JAM-A in the absence and presence of VEGF stimulation (addendum to Fig. 4 C). (B) Quantification of Src phosphorylation (Tyr530) in JAM-A KD MCF7 cells (addendum to Fig. 4 F). Note that Tyr530 of Src is a negative regulatory Tyr residue, and that low pY530/total Src ratios are indicative for high Src activity. (C) Quantification of Erk1/2 phosphorylation (Thr202/Tyr204) in JAM-A KD MCF7 cells (addendum to Fig. 4 G). (D) Quantification of Abi1 phosphorylation (Ser225) in JAM-A KD MCF7 cells (addendum to Fig. 4 H). (E) Quantification of Erk1/2 phosphorylation (Thr202/Tyr204) in JAM-A KD MCF7 cells treated with PP2. (F) Quantification of Abi1 phosphorylation (Ser225) in JAM-A KD MCF7 cells treated with either CI-1040 or with PP2. (G) Quantification of Csk association with CD9 in the absence and presence of VEGF stimulation (addendum to Fig. 5 A). (H) Paxillin localization in MCF7 cells cultured on VN-coated micropatterned substrates (5 µm width). Arrowheads indicate polarized enrichment of paxillin at the periphery of cells migrating on the spatially confined substrate. Scale bars: 10 µm. (I) Paxillin phosphorylation at Tyr118 and Ser126 after inhibition of ERK1/2 (CI-1040) or Src (PP2). Top: Western blot analysis using antibodies against total paxillin (Pax), P-Ser126 paxillin (pS126Pax), or P-Tyr118 paxillin (pY118Pax). Bottom: Quantification of paxillin phosphorylations. Western blot signals were quantified using the Odyssey application software. In A and G, signals obtained with Src and Csk antibodies were normalized to the signals obtained with JAM-A and CD9 antibodies, respectively. In B, C, D, E, F, and I, signals obtained with phospho-specific antibodies were normalized to the signals obtained with antibodies directed against the respective total protein in each experiment. (J) Cell size analysis of MCF7 cells stably transfected with a control vector (pLVTHM-EGFP, Control KD) or with a JAM-A shRNA expression vector (pLVTHM-EGFP-JAM-A-shRNA, JAM-A KD) seeded on VN-coated or FN-coated polyacrylamide gels (for VN: n = 34 for Ctrl cells, n = 35 for JAM-A KD cells, four independent experiments; for FN: n = 74 for Ctrl cells, n = 74 for JAM-A KD cells, four independent experiments). Data is taken from three independent experiments in each panel. Statistical analysis was performed using unpaired Student’s t test. NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Source data are available for this figure: SourceData FS2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal