Fig. 2. The PH domain in Gab1 and PI3K activity are required for Gab1 activation. (A) HEK293T cells were cotransfected with expression vectors for wt-Gab1, Gab1PH or the isolated PH domain, together with vectors encoding EpoR-gp130 chimeric receptors. Two days after transfection, cells were starved in DMEM without FCS for 3 hours and subsequently stimulated with Epo (7 U/ml) for 15 minutes. Cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 to control stimulation of the cells and for the activated forms of ERK1/2 and Gab1. After stripping, the membrane was stained for Gab1 (Flag), STAT3 and ERK to monitor Gab1-transfection efficiency and equal loading of the gels (STAT3, ERK). (B) HEK293T cells were cotransfected with expression vectors for Gab1 and EpoR-gp130 chimeric receptors. Cells were treated as above and then with the PI3K inhibitor LY294002 (40 µM) for 45 minutes prior to stimulation with Epo (7 U/ml) for 15 minutes. After western blotting, the membranes were stained for Gab1 phosphorylated at Y627 and for Akt phosphorylated at Ser473 (pS473Akt) to monitor PI3K activity. After stripping, the membrane was stained for Gab1 and Akt to monitor Gab1-transfection efficiency and equal loading of the gel. The shift in Akt mobility is due to phosphorylation of the protein. Owing to the use of Gab1-GFP fusion proteins in the following experiments and to better distinguish endogenous from exogenous Gab1 proteins, the latter were expressed as GFP-fusion proteins to reduce mobility in SDS-PAGE.