Insulin receptors (IR) in APS−/− mice and culture cells. A: Insulin receptor protein expression in liver, skeletal muscle, heart, brain, and adipose tissue in APS+/+ and APS−/− mice identified by Western blot analysis. Thirty micrograms of total lysate was subjected to SDS-PAGE and blotted with anti-insulin
receptor β-antibody. B: 3T3L1-G4myc-CARΔ1 adipocytes were exposed to adenovirus encoding APS wild-type (WT), Y618F mutant (YF), or GFP as a control
(MOI; 10–20 pfu/cell). Cells were serum starved, then stimulated with (+) or without (−) 100 nmol/l insulin for 5 min, and
lysates were immunoprecipitated (IP) with anti-APS COOH-terminus antibody (5) or anti-Cbl antibody and immunoblotted (IB) with anti-APS NH2-terminus (10), anti-4G10, anti-Cbl, or anti-insulin receptor β-antibodies, as described in research design and methods.