pAkt

Phosphorylated Akt (Akt-pS473) was determined using an Enzyme-linked immunosorbent assay (ELISA) kit, following the recommendation of the manufacturer (Elabscience, China).

Confluent cultures of C2C12 skeletal muscle cells grown in a 6 well-plates were treated with 1 mg/ml of specific extracts and insulin solution (100 nM) for 3 h. The cells were repeatedly (3X) washed with pre-warmed PBS and lysed by the addition of cell lysis buffer in which protease and phosphate inhibitors were added. The lysates were suspended and incubated with shaking at 8 ^oC for 30 min and then centrifuged at 13000 rpm for 10 min at 8 ^oC. The supernatant transferred to clean test tube and used immediately or stored at -70 ^oC. A volume of 100 µl of each sample or positive control was added into appropriate wells, which were covered with a plate holder and incubated overnight at 4 ^oC with shaking. The solution was subsequently discarded and the wells washed 4 times with 1 x wash solution. The plate was inverted and blotted against a clean paper towels. An amount of 100 µl of prepared 1000 dilution of rabbit anti-phospho-Akt (Ser473) antibody was added to each well, followed by incubation for 1 h at room temperature with shaking. The solution was then discarded and the wells washed 4 times with wash buffer, followed by an addition of a volume of 100 µl of prepared 1 x horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG to each well and incubated for 1 h at room temperature with shaking. The solution was again discarded and the wells washed 4 times with washed buffer. A volume of 100 µl of TMB one–step substrate reagent was subsequently added to each well and incubated for 30 minutes at room temperature with shaking in the dark. Then, 50 µl of stop solution was added to each well and the plate was read immediately at 450 nm using a microplate reader.