Figure 4

Interaction of Gab1 with N-SH2 domain of p85 subunit of PI3K. Retinal proteins from (two independent rats) controls and H₂O₂ (600 μM) treated ex vivo retinal explants were subjected to GST pull-down assay with N-SH2 domain of p85, followed by immunoblot analysis with anti-Gab1 antibody (A). The blot was reported with GST to ensure equal amount of fusion in each lane (A). PTP1B dephosphorylates the tyrosine phosphorylation of IR in vitro . Rat retinas were dissected and incubated at 37°C for 5 min in DMEM medium in the presence or absence of insulin (1 μM). After incubation, the retinas were lysed and subjected to immunoprecipitation with anti-IRβ antibody. The anti-IRβ immune complexes were subjected to in vitro dephosphorylation by PTP1B in the presence or absence of H₂O₂ for 15 min at 37°C. The reaction after dephosphorylation was subjected to immunoblot analysis with anti-PY99 antibody (B). The blot was stripped and reprobed with anti-IRβ antibody to ensure equal amounts of protein in each immunoprecipitate (B). Inhibition of PTP1B activity in light-adapted retina. Retinas from each rat were immunoprecipitated with anti-PTP1B antibody and PTP1B activity measured (D). Data are mean ± SD, n=5, *p<0.05. Twenty μg of retinal proteins from dark- and light-adapted rat retinas (two independent rats) were immunoblotted with anti-PTP1B and anti-actin antibodies (C).