Cells were lysed in RIPA buffer and protein concentration was measure by BCA assay (Thermo Scientific). Aliquots with equal protein amounts were suspended in SDS PAGE loading buffer and run in 140V for 1hr. Protein was transferred onto a nitrocellulose membrane (Bio-Rad) with 100V for 1hr in 4°C. Membranes were blocked with 5% milk in 1hr then incubated with primary antibody overnight in 4°C. Primary antibodies included Anti-Akt1, Anti-pAkt(S473), Anti-pAkt(T308), Anti-pGSK3b, Anti-pPTEN, GAPDH, Anti-pPDK1, from Cell Signaling, and Anti-Akt1from Thermo Scientific. Actin was obtained from Secondary antibodies: Anti-Rabbit IgG from Novus Biologicals (Littleton, CO).
Membranes were then washed and incubated with secondary antibody for 1hr at room temperature. Membranes were washed and incubated in Western blot substrate (Thermo Scientific) for 2 min. Membranes were exposed on X-ray film.
Membranes were then washed and incubated with secondary antibody for 1hr at room temperature. Membranes were washed and incubated in Western blot substrate (Thermo Scientific) for 2 min. Membranes were exposed on X-ray film.