FIG. 6.

Dose-dependent effects of ectopic APS expression on insulin-stimulated Cbl phosphorylation. 3T3-L1 adipocytes were cotransfected with various amount of vector DNA and Myc-tagged wild-type APS (Myc-APS/WT) or Myc-APS/Y⁶¹⁸F. Lanes 1 and 6, 300 μg of vector plus 0 μg of APS; lanes 2 and 7, 285 μg of vector plus 15 μg of APS; lanes 3 and 8, 270 μg of vector plus 30 μg of APS; lanes 4 and 9, 200 μg of vector plus 100 μg of APS; lanes 5 and 10, 0 μg of vector and 300 μg of APS. 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 antibody (lower panels).