EPO-induced phosphorylation of Gab2, but not Gab1, is dependent on PI3K activity. (A) Erythroblast cell line R1 was factor deprived (4 hours), left untreated (lane 1), or restimulated with EPO (5 U/mL; 10 minutes) in the presence of increasing amounts of the PI3K inhibitor LY294002. Gab1 and Gab2 were immunoprecipitated from cell lysates and analyzed for tyrosine phosphorylation on Western blot. The same blots were stained with anti-Gab1 and anti-Gab2 to control equal loading. Coimmunoprecipitated Shp2 and Shc were detected by specific antibodies. Size markers (kDa) and coimmunoprecipitating proteins are indicated next to the panels. (B) Tyrosine phosphorylation of Gab1 and Gab2 in the presence of increasing concentrations of LY294002 was quantified by densitometric analysis as shown in panel A. Intensity in absence of LY294002 was set at 100%.