Figure 7

Interaction of wild type and phosphorylation deficient mutants of Gab1 with substrate trapping mutant of PTP1B. Myc-tagged wild type and various phosphorylation deficient mutants (Y83F; Y285F; Y373F; Y407F; Y448F; Y473F; Y590F; Y628F; Y660F) were expressed in HEK-293 T cells followed by pervanadate treatment of cells as described in methods. Proteins from wild type and various mutants of Gab1 were incubated with either wild type or mutant PTP1B followed by GST pull-down assay and the bound proteins were subjected to 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). Denistomeric analysis of immunoblots was performed in the linear range of detection and the binding of various tyrosine mutants of Gab1 to mutant PTP1B was normalized to their respective loading control (lysate) (B). The wild type Gab1 binding to mutant PTP1B was set as 100% (B).