Phosphorylated akt at s473

Cells were rinsed and covered with PBS, scraped off the plate, and spun down, the liquid was aspirated, and the cells were frozen in liquid nitrogen and stored at –80°C. Cells were then thawed on ice and lysed (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, and 0/1% SDS in water + protease inhibitor cocktail [Sigma] + phosphatase inhibitor cocktail [Sigma-Aldrich] + okadaic acid + sodium fluoride). Sample protein concentrations were measured with a Bradford assay (Bio-Rad) and equal protein concentrations were run on NuPAGE 4–2% Bis–Tris gels (Invitrogen). Protein was transferred to a nitrocellulose membrane and incubated in blocking solution (PBS, 5% BSA, 0.1% Tween 20) for 30 min at room temperature. The membrane was incubated with primary antibodies in blocking solution at 4°C overnight, rinsed three times in PBS with 0.1% Tween 20, and then incubated in secondary antibody for 1 h at room temperature. Antibodies: total AKT (Cell Signaling), phosphorylated AKT at S473 (Cell Signaling), 680LT secondary (LICOR-IRDye), 800CW secondary (LICOR-IRDye), actin (Sigma). The blot was imaged on the LI-COR Odyssey.