P akt 473

Total proteins were lysed with lysis buffer supplemented with 1% PMSF, and the concentration was determined using a BCA protein assay kit (Sigma, St Louis, MO, USA). Cell lysates were loaded on 12% polyacrylamide gels and subsequently transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% milk and incubated with antibodies against PTEN (1/1,000 diluted; Proteintech, USA), p-Akt 308, p-Akt 473 and Akt (1:1,000 diluted; abgent, San Diego) at 4 °C overnight, followed by incubation with anti-rabbit IgG (1/5000 diluted; GE Healthcare UK Ltd., Little Chalf-ont, UK) at 37 °C for 2 h. GAPDH antibody (1/2500 diluted; Bioworld) was used as an internal standard to normalize protein expression. All bands were detected using ECL Western blot kit (Amersham Biosciences, UK), according to the manufecturer’s protocol.

The extracted MCF-7 and MDA-MB-231 cells were split using cell lysates (KeyGEN Company) with protease inhibitor cocktail (Biotool, Houston, TX, USA). Whole-cell proteins were electrophoresed under the conditions in 12% polyacrylamide gels. The separated proteins were transferred to a polyvinylidenedifluoride membrane. Nonspecific binding was blocked with 5% skimmed milk for 2 h at 37 °C. The membrane was then incubated with PTEN (1 : 1500 diluted; Proteintech), p-Akt 308, p-Akt 473 and Akt antibody (1 : 1000 diluted; Abgent, San Diego, CA, USA) at 4 °C overnight. A GAPDH antibody (1 : 2500 diluted; Bioworld, Minneapolis, MN, USA) was used as a control. Next, the membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1 : 5000 diluted; Thermo, Boston, MA, USA). Images were obtained from multifunctional gel imaging system (ImageQuant LAS 500, General Electric, Fairfield, CT, USA).