Untreated (–) and treated (+) NIH-3T3 whole cell extracts (30 μg) were separated by 10% SDS-PAGE, and the membrane was blotted with AKT (phospho Ser473) antibody [HL2971] (GTX640358) diluted at 1:1000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody.

Specificity test analysis was performed by coating the plate with recombinant AKT1 peptide and AKT1 (phospho Ser473) peptide (1000-15.63 ng/mL). Coated protein was probed with AKT (phospho Ser473) antibody [HL2971] (GTX640358) (1 μg/mL). Goat anti-rabbit IgG antibody (HRP) (GTX213110-01) (1:10000) was used to detect the bound primary antibody.