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. 2020 Aug 26;9:e57617. doi: 10.7554/eLife.57617

Figure 6. SHANK2 R240 methylation activated endosomal FAK signals.

(A) Phosphorylated/total FAK and cortactin were determined in MCF-7 cells expressing the indicated Flag-tagged PRMT7. Western blot was performed with the indicated antibodies. (B) Phosphorylated/total FAK and cortactin were determined in MDA-MB-231 cells with or without stable expression of the indicated SHANK2 shRNA or a control shRNA. Western blot was performed with the indicated antibodies. (C) Phosphorylated/total FAK and cortactin were determined in MDA-MB-231 cells with or without expressing SHANK2 shRNA and with or without reconstituted expression of WT SHANK2 or SHANK2 R240K mutant. Western blot was performed with the indicated antibodies. (D) Phosphorylated/total FAK and cortactin were determined in MDA-MB-231 cells expressing SHANK2 shRNA with or without reconstituted expression of WT SHANK2 or SHANK2 R240K or SHANK2 R240F mutant, SHANK2 R240F were treated with GSK2256098 (10 µM) or DMSO as control. Western blot was performed with the indicated antibodies. (E) Phosphorylated/total FAK and cortactin were determined in MCF10A cells co-transfected with SHANK2 and/or FAK plasmids. Western blot was performed with the indicated antibodies. (F) Phosphorylated/total FAK and cortactin were determined in MDA-MB-231 cells expressing SHANK2 shRNA with or without reconstituted expression of WT SHANK2, SHANK2 R240K or SHANK2 R240F mutant. SHANK2 R240F group was treated with dynasore (80 µM) or DMSO as control. Western blot was performed with the indicated antibodies. (G) Phosphorylated/total FAK and cortactin were determined in MDA-MB-231 cells expressing SHANK2 shRNA with or without reconstituted expression of WT SHANK2, SHANK2 R240K or SHANK2 R240F mutant. SHANK2 R240F with or without stable expression of the indicated EEA1 shRNA or a control shRNA. Western blot was performed with the indicated antibodies.

Figure 6.

Figure 6—figure supplement 1. SHANK2 R240F activated FAK and cortactin.

Figure 6—figure supplement 1.

(A) Phosphorylated/total FAK and cortactin were determined in MCF10A cells co-transfected with SHANK2 and/or cortactin plasmids. Western blot was performed with the indicated antibodies. (B) MCF10A cells co-transfected with SHANK2 and/or cortactin plasmids. Assessment of MMP9 enzymatic activities by gelatin zymography. (C) MCF10A cells co-transfected with SHANK2 and/or cortactin plasmids. Assessment of the MMP9 enzymatic activities by gelatin zymography. (D) MDA-MB-231 cells with or without expressing SHANK2 shRNA and with or without reconstituted expression of SHANK2 WT or SHANK2 R240K mutant. Flow cytometric analysis of annexin V-FITC/PI staining was conducted to examine cell death.