The Y731F mutant of c-Cbl cannot associate with p85 by IL-4 stimulation. (A) The establishment of YF cells, which stably express Y731F mutant of c-Cbl. Lysates from mock, HCbl, and YF cells were immunoprecipitated with anti–c-Cbl antibody and subjected to the immunoblotting with anti-HA antibody, 12CA5. The construct of Y731F mutant of c-Cbl is presented in Fig 3A. An arrow indicates c-Cbl. (B) Y731F c-Cbl mutant cannot associate with p85. Lysates from HCbl and YF stimulated with IL-4 (10 ng/mL) for 5 minutes at 37°C were immunoprecipitated with anti-p85 antibody, followed by the immunoblotting with antiphosphotyrosine antibody (upper panel) or anti-p85 antibody (lower panel). An arrow indicates c-Cbl.