Gab1 is an interaction partner and a substrate of RON in erythroblasts. (A) Immortalized (R1) and primary fetal liver-derived erythroblasts were factor deprived and subsequently stimulated with EPO (5 U/mL; 10 minutes) or MSP (100 ng/mL; 10 minutes). Anti-RON immunoprecipitates were analyzed for proteins phosphorylated on tyrosine using monoclonal PY99. Blots were stained with anti-RON to control phosphorylated protein comigration with RON and to control loading. Arrows indicate the position of RON and the position plus molecular weight of phosphorylated coimmunoprecipitated proteins. Anti-RON did not stain a 150-kDa protein in immunoprecipitates obtained with unrelated antibodies (eg, anti-Btk; data not shown), indicating that the 150-kDa band is specific for the anti-RON immunoprecipitate. (B) HEK293 cells were mock transfected or transfected with pSG5-RONfl, serum deprived, and stimulated with EPO (5 U/mL; 10 minutes) or MSP (100 ng/mL; 10 minutes). Anti-RON immunoprecipitates were tested for RON phosphorylation, as described for panel A. (C-D) Erythroblast cell line R1 (C) and fetal liver-derived erythroblasts (D) were factor deprived and restimulated with EPO (5 U/mL; 10 minutes). Gab1 was immunoprecipitated, and blots were stained with antiphosphotyrosine antibodies (PY99) and antibodies against Gab1 and RON, as indicated. (E-F) Fetal liver-derived erythroblasts derived from RON TK-/-embryos and wt littermates were factor deprived and restimulated with EPO (5 U/mL; 10 minutes). Western blots containing Gab1 and Gab2 immunoprecipitates were stained with antiphosphotyrosine and with anti-Gab1 or anti-Gab2 to check equal loading.