Horseradish peroxidase hrp conjugated goat antimouse igg

Proteins were extracted from the tissue samples using the TRI reagent, according to the manufacturer's protocol. Equal amounts of each protein sample (30 μg) were subjected to SDS PAGE and transferred to PVDF membranes. Membranes were blocked with a blocking buffer containing 10% skim milk (Millipore, USA) and 5% TBST. The membranes were probed with primary antibodies including anti-Akt antibody (1: 2000, ab30471, Elabscience, USA), anti-p-Akt (Thr 308) antibody (1 : 1000, sc271966, Santa Cruz, USA), anti-β actin antibody (1 : 1000, sc47778, Santa Cruz, USA), antiglycogen synthase 2 antibody (1 : 1000, sc390391, Santa Cruz, USA), and anti-phospho-glycogen synthase (Ser641) antibody (1 : 1000, Cell Signaling, USA) for 1.5 hours at room temperature. Subsequently, the membranes were incubated for 1 hour at room temperature with an appropriate secondary antibody: horseradish peroxidase- (HRP-) conjugated goat antimouse IgG (1 : 5000, Dako, P0447) or HRP-conjugated goat antirabbit IgG (1 : 16000, Santa Cruz, SC2301). Bands were visualized by an Amersham ECL Advance Western Blotting Detection Kit (GE healthcare). The activity of AKT protein was measured by comparing the level of phosphorylated AKT with the level of nonphosphorylated AKT. The ImageJ software (National Institutes of Health, Bethesda, MD, USA) was utilized for quantification of the intensity band.