FIG. 2.
Association with APS is necessary for the tyrosine phosphorylation of Cbl by the insulin receptor. CHO-IR cells (A) or 3T3-L1 adipocytes (B) were cotransfected with HA-Cbl and vector DNA, Myc-tagged wild-type APS (Myc-APS/WT) or APS/Y618F. Separately, CHO-IR cells were also cotransfected with HA-Cbl and empty vector or FLAG-CAP (C). After treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed using an anti-HA antibody. Immunoprecipitates were analyzed by antiphosphotyrosine, anti-HA, anti-Myc, and anti-Crk immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc, anti-FLAG, anti-HA, and anti-phospho-ERK antibodies (lower panels).