Distinct regulation of Gab1 and Gab2 tyrosine phosphorylation. (A-C) COS cells were transfected with pSG5-based expression constructs encoding Gab1, Gab2, and various kinases, as indicated. After 48 hours, cells were harvested. (A) Phosphorylation status of transfected kinases was detected on Western blots using antiphosphotyrosine antibodies (PY99). Similarly, Gab1 (B) and Gab2 (C) immunoprecipitates were analyzed for tyrosine phosphorylation. Arrows indicate the positions of Gab1, Gab2, RONsf, and RONfl. Size markers are indicated in kDa. Blots were restained with anti-Gab1 or anti-Gab2, as indicated. (D-E) R1 erythroblasts (D) and fetal liver-derived erythroblasts (E) were factor deprived for 4 hours in the absence or presence of the Src kinase family inhibitor PP2 (5 μM), as indicated. Cells were left unstimulated or were stimulated with EPO (5 U/mL; 10 minutes). EPO-induced Gab1 or Gab2 phosphorylation was assayed on immunoblots using PY99, and equal loading was controlled by staining with anti-Gab1 or anti-Gab2. Size markers are indicated in kDa. (F) Gab 1 associated directly with RON in Y2H assays. Gab proteins were coexpressed with RON, RONkd, and Grb2 in the yeast reporter strain L40. Gab2+MBS encodes Gab2 protein with an insertion of MBS. Associations were quantified by β-galactosidase liquid assay.