Figure 5. PTP-PEST forms a multi-protein complex with Cas and Vcp in GBM cells.

(A); Pull-down experiments with GST-tagged PTP-PEST proteins (WT and C/S) reveal interactions with Vcp and Cas in detergent-soluble GSC lysates. (B, C); Lysates from GSC6-27 control or PTPN12 KO-1 cell pools were immunoprecipitated with anti-PTP-PEST antibodies and resolved lysates were immunoblotted to analyze protein-protein interactions. Note that Cas (B) and Vcp (C) proteins interact in control lysates but not in PTPN12 KO lysates as revealed by co-immunoprecipitation. (D, E); Detergent-soluble lysates from GSC6-27 control or PTPN12 KO-1 cells were immunoprecipitated with anti-Vcp (D) or anti-Cas (E) antibodies and resolved lysates were immunoblotted to analyze protein-protein interactions. (F); Tyrosine phosphorylated Cas protein or total cellular phosphotyrosine-containing proteins were immunoprecipitated from control or PTPN12 KO GSC6-27 cells and Vcp interactions were analyzed by immunoblotting with anti-Vcp antibodies. Note that in comparison to control cells, Vcp is hyperphosphorylated in PTPN12 KO-1 cells, but also shows reduced interactions with phosphorylated Cas. (G); Detergent-soluble lysates from control (n=2) and PTPN12 KO GSC6-27 cells (n=2) were immunoblotted for phosphorylated Cas and other phospho-specific antibodies recognizing common focal adhesion proteins such as FAK and paxillin. (H); Control and PTPN12 KO GSC6-27 cell lysates were immunoblotted for total EGFR, total Her2/ErbB2, or specific cytoplasmic domain phosphorylated sites on each protein. Note the increased levels of EGFR and Her2/ErbB2 tyrosine phosphorylation in PTPN12 KO GSCs.