Fig. 5. Activation of the MAP kinase pathway by Src^527F and Src^527F/c-Yes chimeras. (A) 500 µg of RIPA lysates from mock-transfected CEF or cells expressing Src^527F, Y4U32^527F or Y4U^527F were immunoprecipitated with an anti-Shc antibody and separated by 10% SDS PAGE. Immunoprecipitated proteins were transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T and probed with an anti-Grb2 antibody (top panel). The blots were stripped and re-probed with the anti-Shc antibody, immunoreactive against the 46 and 52 kDa isoforms of Shc (bottom panel). (B) 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src^527F, Y4U32^527F, or Y4U^527F were resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T and probed with an anti-phospho-Raf antibody (top panel). Western blot analysis was also performed with an anti-c-Raf antibody to determine relative protein levels (bottom panel). (C) 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src^527F, Y4U32^527F or Y4U^527F were processed for western blot analysis as described above. Membranes were probed with an anti-phospho-Erk antibody (top panel) or an anti-p42/44 MAPK antibody to determine relative protein levels (bottom panel). (A-C) Lane 1, CEF; lane 2, Src^527F; lane 3, Y4U32^527F; lane 4, Y4U^527F.