Fig. 4
PIK3R1 loss alters phosphorylation of Gab2 at S210 and S623, which in turn leads to signaling activation. a SKOV3, OVCAR8, and OAW28 cells were transfected with siRNA for 72 h prior to western blotting. b Protein lysates of HEK-293T transfected with PIK3R1 siRNA or non-specific siRNA were subjected to immunoprecipitation with Gab2 antibody. The immunoprecipitates were subjected to SDS-PAGE separation for mass spectrometry-based quantitative phosphoproteomics. The MS/MS spectrum of phosphopeptide containing the S210 phosphorylated residue of Gab2 is shown. Inset, the log2 intensities of S210 phosphorylation in the samples. c SKOV3, OVCAR8, and OAW28 cells were transfected with siRNA for 72 h prior to western blotting. d OVCAR5 cells transfected with wild-type GAB2 or GAB2 mutants with or without PIK3R1 siRNA for 72 h were harvested for western blotting. e OVCAR5 cells transfected with HA-tagged wild-type GAB2 or GAB2 S623A mutant were subjected to immunoprecipitation with HA antibody and western blotting. Cells transfected with empty vector but not HA-tagged Gab2 was used as control (labeled as mock). The numbers below the blots represent the mean values from densitometry readings of three independent experiments
