Figure 5.
Csk directly interacts with Tyr280-phosphorylated JAM-A and regulates CIL in MCF7 cells. (A) CD9 IPs from VEGF-stimulated MCF7 cells were blotted for Csk (top panel, 90%) or for CD9 (bottom panel, 10%). Quantification of Western blot signals is shown in Fig. S2 G. (B) Left: In vitro phosphorylation of GST-JAM-A fusion proteins by c-Src. Phosphorylation by aPKCζ served as a positive control. Right: In vitro phosphorylation of JAM-A mutants by recombinant c-Src. (C) Flag-tagged JAM-A constructs (JAM-A/WT, JAM-A/Y280F, and JAM-A/Y261F) were immunoprecipitated from HEK293T cells and analyzed by Western blotting with a P-Tyr280 JAM-A antibody (Rockland #600-401-GN5, top, 90% of input) or with a Flag tag antibody (bottom, 10% of input). (D) Biotinylated JAM-A cytoplasmic domain (Cyt) peptides were incubated with in vitro translated recombinant Csk full length (Csk/f.l.) or Csk SH2 domain (Csk/SH2). Abbreviations: Pept-PD, peptide pulldown. (E) JAM-A immunoprecipitates obtained from HEK293T cells transfected with JAM-A, Csk, and constitutively active Src (Src/Y530F) were immunoblotted with antibodies against Csk (80% of input), JAM-A (10% of input), and phosphotyrosine residues (4G10, 10% of input). (F) Quantification of CIL phenotypes in Csk KD MCF7 cells. Collisions of control MCF7 cells (transfected with pTRIPZ-tRFP vector, Ctrl KD) or Csk KD MCF7 cells (transfected with pTRIPZ-tRFP-Csk-shRNA, Csk KD) with MCF7 WT cells (transfected with pLVTHM-EGFP). Number of collisions analyzed: n = 122 cells for Ctrl KD–WT collisions (three independent experiments), n = 153 for Csk KD–WT collisions (four independent experiments). (G) Tyr280 phosphorylation of JAM-A was analyzed by immunoprecipitating P-Tyr280-phosphorylated JAM-A from VEGF-stimulated MCF7 cells using a P-Tyr280-JAM-A (P-JAM-A)-specific antibody (Affi1550), followed by immunoblotting with a JAM-A-specific mouse mAb (BD TL 612120). Postnuclear supernatants (PNS) were immunoblotted with a JAM-A antibody (mouse mAb BD TL 612120). Western blot signals were quantified using the Odyssey imaging system (LI-COR). P-Tyr280 JAM-A signals were normalized to total JAM-A levels. Signals obtained from control samples were set to 100%. Statistical analyses were performed using one-sample t test (five independent experiments) NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Quantitative analysis of CIL types observed in 1D kinematic assays performed with mixed populations of WT MCF7 cells and MCF7 cells with doxycycline-inducible KD of JAM-A (pEmU6proT plasmid; − dox: shRNA expression off, + dox: shRNA expression on) and ectopic expression of shRNA-resistant murine JAM-A constructs (mJAM-A/WT, mJAM-A/Y281F). Number of events analyzed: − dox: 121 (3 ind. exp.), + dox: 117 (three independent experiments), + dox:mJAM-A/WT: 120 (four independent experiments), + dox:mJAM-A/Y281F: 89 (four independent experiments). If not indicated otherwise, statistical analyses were performed using unpaired Student’s t test. Data are presented as arithmetic means ± SD; NS, not significant; *, P < 0.05; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.

Csk directly interacts with Tyr280-phosphorylated JAM-A and regulates CIL in MCF7 cells. (A) CD9 IPs from VEGF-stimulated MCF7 cells were blotted for Csk (top panel, 90%) or for CD9 (bottom panel, 10%). Quantification of Western blot signals is shown in Fig. S2 G. (B) Left: In vitro phosphorylation of GST-JAM-A fusion proteins by c-Src. Phosphorylation by aPKCζ served as a positive control. Right: In vitro phosphorylation of JAM-A mutants by recombinant c-Src. (C) Flag-tagged JAM-A constructs (JAM-A/WT, JAM-A/Y280F, and JAM-A/Y261F) were immunoprecipitated from HEK293T cells and analyzed by Western blotting with a P-Tyr280 JAM-A antibody (Rockland #600-401-GN5, top, 90% of input) or with a Flag tag antibody (bottom, 10% of input). (D) Biotinylated JAM-A cytoplasmic domain (Cyt) peptides were incubated with in vitro translated recombinant Csk full length (Csk/f.l.) or Csk SH2 domain (Csk/SH2). Abbreviations: Pept-PD, peptide pulldown. (E) JAM-A immunoprecipitates obtained from HEK293T cells transfected with JAM-A, Csk, and constitutively active Src (Src/Y530F) were immunoblotted with antibodies against Csk (80% of input), JAM-A (10% of input), and phosphotyrosine residues (4G10, 10% of input). (F) Quantification of CIL phenotypes in Csk KD MCF7 cells. Collisions of control MCF7 cells (transfected with pTRIPZ-tRFP vector, Ctrl KD) or Csk KD MCF7 cells (transfected with pTRIPZ-tRFP-Csk-shRNA, Csk KD) with MCF7 WT cells (transfected with pLVTHM-EGFP). Number of collisions analyzed: n = 122 cells for Ctrl KD–WT collisions (three independent experiments), n = 153 for Csk KD–WT collisions (four independent experiments). (G) Tyr280 phosphorylation of JAM-A was analyzed by immunoprecipitating P-Tyr280-phosphorylated JAM-A from VEGF-stimulated MCF7 cells using a P-Tyr280-JAM-A (P-JAM-A)-specific antibody (Affi1550), followed by immunoblotting with a JAM-A-specific mouse mAb (BD TL 612120). Postnuclear supernatants (PNS) were immunoblotted with a JAM-A antibody (mouse mAb BD TL 612120). Western blot signals were quantified using the Odyssey imaging system (LI-COR). P-Tyr280 JAM-A signals were normalized to total JAM-A levels. Signals obtained from control samples were set to 100%. Statistical analyses were performed using one-sample t test (five independent experiments) NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Quantitative analysis of CIL types observed in 1D kinematic assays performed with mixed populations of WT MCF7 cells and MCF7 cells with doxycycline-inducible KD of JAM-A (pEmU6proT plasmid; − dox: shRNA expression off, + dox: shRNA expression on) and ectopic expression of shRNA-resistant murine JAM-A constructs (mJAM-A/WT, mJAM-A/Y281F). Number of events analyzed: − dox: 121 (3 ind. exp.), + dox: 117 (three independent experiments), + dox:mJAM-A/WT: 120 (four independent experiments), + dox:mJAM-A/Y281F: 89 (four independent experiments). If not indicated otherwise, statistical analyses were performed using unpaired Student’s t test. Data are presented as arithmetic means ± SD; NS, not significant; *, P < 0.05; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.

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