FIG. 5.

Overexpression of APS blocks insulin-stimulated tyrosine phosphorylation of Cbl and Crk-Cbl interaction. 3T3-L1 adipocytes were transfected with vector alone, Myc-tagged wild-type APS (Myc-APS/WT), or Myc-APS/Y⁶¹⁸F. (A) Following treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed with an anti-Cbl antibody. Immunoprecipitates were analyzed by antiphosphotyrosine and anti-Cbl immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc and anti-phospho-ERK antibodies (lower panels). (B) Following treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation was performed with an anti-Crk antibody. Immunoprecipitates were analyzed by anti-Cbl and anti-Crk immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc, anti-Cbl, and anti-phospho-ERK antibodies (lower panels).