Figure 8

Interaction of various mutants and deletion constructs of Gab1 with substrate trapping mutant of PTP1B. Wild type Myc-tagged Gab1, Myc-Gab1 (Y47F), Myc-Gab1 (K49T), Myc-Gab1 (K49A), Myc-Gab1 (ΔEYYK) and truncated version of Myc-Gab1 (1–280 amino acids) constructs were expressed in HEK-293 T cells followed by pervanadate treatment. Pervanadate treated samples were subjected to GST pull-down assay with either wild type PTP1B or mutant PTP1B followed by immunoblot analysis with anti-Myc antibody (A). The blot was reprobed with anti-GST antibody to ensure equal amount of fusion in each pull-down (A). PH domain is necessary for the interaction of Gab1 with substrate trapping mutant of PTP1B. Myc-tagged Δ PH-Gab1 was expressed in HEK-293 T cells followed by pervanadate treatment. Pervanadate treated samples were subjected to GST pull-down assay with either wild type PTP1B or mutant PTP1B followed by immunoblot analysis with anti-Myc antibody (B). The blot was reprobed with anti-GST antibody to ensure equal amount of fusion in each pull-down (B). Proteins from wild type and Δ PH-Gab1 was subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-pGab1 antibody (B, bottom panel).