Figure 5

Identification of Gab1 as PTP1B substrate with substrate trapping mutant technique. Pervanadate-treated retinal ex vivo explants or Gab1 expressed HEK-293 T cell lysates were subjected to GST pull-down assay with either wild type PTP1B or mutant PTP1B-D181A followed by immuno blot analysis with anti-Gab1 antibody (A). The blot was reprobed with anti-GST antibody to ensure equal amounts of fusion in each pull-down (A). Lysate, retinal proteins and HEK-293 T cell expressed Gab1 were used as positive controls. Co-localization of D181A-PTP1B and Gab1. HEK-293 T cells cotransfected with Myc-tagged D181A-PTP1B and Gab1 expression plasmids were fixed with paraformaldehyde and processed for immunofluorescence and visualized by confocal microscopy. Cells were incubated with anti-Myc (red) and Gab1 (green) antibodies (B). Overexpressed PTP1B and Gab1 were visualized with fluorescein-conjugated sheep anti-mouse and Texas red-conjugated goat anti-rabbit antibodies, respectively. Right panel represents the merge image of D181A-PTP1B and Gab1. PTP1B dephosphorylates the Gab1 tyrosine phosphorylation in vitro. Myc-tagged Gab1 was transfected (four independent transfections) into HEK-293 T cells and the proteins were subjected to immunoprecipitation with anti-Myc antibody. The immune complexes were incubated with GST fusion proteins of either wild type PTP1B or catalytically inactive PTP1B (D181A) for 15 min at 30°C. At the end of the reaction, the immune complexes were subjected to SDS-PAGE followed by immunoblot analysis with anti-PY99 antibody (C). The blot was stripped and reprobed with anti-Myc and anti-GST antibodies (C).