Figure 4.
JAM-A interacts with CD9, αvβ5 integrin, and c-Src, and suppresses the activities of the Src-FAK and MEK-ERK1/2 pathways. (A) CoIP of αvβ5 integrin with JAM-A. (B) CoIP of CD9 with JAM-A. (C) CoIP of Src with JAM-A. Src association with JAM-A is only detectable in Brij98 lysates. Quantification of Western blot signals is shown in Fig. S2 A. (D) CoIP of Src with CD9. (E) CoIP of αvβ5 integrin with Src. (F) CD9 immunoprecipitates from control MCF7 cells (MCF7-pLVTHM, left panel) or JAM-A-depleted cells (MCF7-pLVTHM-JAM-A shRNA, right panel) were immunoblotted for Tyr530-phosphorylated Src (80% of input, top panels). Note that high Src activity is reflected by low P-Tyr530 immunoreactivity. About 10% of the precipitated material was immunoblotted for total Src and for CD9. Data are representative of three independent experiments. Quantifications of Western blot signals are shown in Fig. S2 B. (G) Lysates of control MCF7 cells or JAM-A KD MCF7 cells were immunoblotted for P-Thr202/P-Tyr204 ERK1/2 or total ERK1/2. Data is representative of three independent experiments. Quantifications of Western blot signals are shown in Fig. S2 C. (H) Abi1 immunoprecipitates were immunoblotted for Ser225-phosphorylated Abi1 (90% of input, top panels) and total Abi1 (10% of input, bottom panels). In the bottom panel, an Abi1 immunoprecipitate obtained from unstimulated control cells (lane 3) was loaded directly adjacent to an Abi1 immunoprecipitate obtained from unstimulated JAM-A-depleted cells (lane 4) for direct comparison of Abi1 P-Ser225 levels. Quantifications are shown in Fig. S2 D. (I) Quantification of CIL types after collisions between scrambled shRNA-expressing MCF7 cells (Ctrl KD) and JAM-A shRNA-expressing MCF7 cells (JAM-A KD) in the presence of either CI-1040 or PP2. Number of collisions analyzed: n = 111 for Ctrl KD–WT collisions, n = 112 for JAM-A KD–WT collisions, n = 81 for JAM-A KD—WT (+CI-1040), n = 61 for JAM-A KD—WT (+PP2; three independent experiments). Statistical analysis was performed with unpaired Student’s t test. Data are presented as mean values ± SD. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F4.

JAM-A interacts with CD9, αvβ5 integrin, and c-Src, and suppresses the activities of the Src-FAK and MEK-ERK1/2 pathways. (A) CoIP of αvβ5 integrin with JAM-A. (B) CoIP of CD9 with JAM-A. (C) CoIP of Src with JAM-A. Src association with JAM-A is only detectable in Brij98 lysates. Quantification of Western blot signals is shown in Fig. S2 A. (D) CoIP of Src with CD9. (E) CoIP of αvβ5 integrin with Src. (F) CD9 immunoprecipitates from control MCF7 cells (MCF7-pLVTHM, left panel) or JAM-A-depleted cells (MCF7-pLVTHM-JAM-A shRNA, right panel) were immunoblotted for Tyr530-phosphorylated Src (80% of input, top panels). Note that high Src activity is reflected by low P-Tyr530 immunoreactivity. About 10% of the precipitated material was immunoblotted for total Src and for CD9. Data are representative of three independent experiments. Quantifications of Western blot signals are shown in Fig. S2 B. (G) Lysates of control MCF7 cells or JAM-A KD MCF7 cells were immunoblotted for P-Thr202/P-Tyr204 ERK1/2 or total ERK1/2. Data is representative of three independent experiments. Quantifications of Western blot signals are shown in Fig. S2 C. (H) Abi1 immunoprecipitates were immunoblotted for Ser225-phosphorylated Abi1 (90% of input, top panels) and total Abi1 (10% of input, bottom panels). In the bottom panel, an Abi1 immunoprecipitate obtained from unstimulated control cells (lane 3) was loaded directly adjacent to an Abi1 immunoprecipitate obtained from unstimulated JAM-A-depleted cells (lane 4) for direct comparison of Abi1 P-Ser225 levels. Quantifications are shown in Fig. S2 D. (I) Quantification of CIL types after collisions between scrambled shRNA-expressing MCF7 cells (Ctrl KD) and JAM-A shRNA-expressing MCF7 cells (JAM-A KD) in the presence of either CI-1040 or PP2. Number of collisions analyzed: n = 111 for Ctrl KD–WT collisions, n = 112 for JAM-A KD–WT collisions, n = 81 for JAM-A KD—WT (+CI-1040), n = 61 for JAM-A KD—WT (+PP2; three independent experiments). Statistical analysis was performed with unpaired Student’s t test. Data are presented as mean values ± SD. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F4.

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