Figure 2. Liver insulin signaling cascade total proteins and phospho-proteins in response to insulin stimulation (10 IU/kg) versus saline treated controls in GHRKO (KO) and normal (N) mice fed ad libitum (AL) or 30% calorie restricted (CR) for one year.
ELISA with antibodies directed towards total insulin receptor (IR; A), IR pY1158 (B), AKT1 (D), and AKT1 pS473 (E) were performed. Liver homogenates were immunoprecipitated (IP) with anti-p85, separated using SDS-PAGE and the level of non-specific Tyr phosphorylation was determined at approximately 180 kDa using anti-pY99 (C). Total AKT2 protein (F) and AKT2 pS473/474 (G) was determined from liver protein homogenates first subjected to IP using anti-AKT2. Panel (H) summarizes key insulin-stimulated phospho-proteins compared to insulin-stimulated N AL mice. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05).