Sab4504331

Proteins (15–20 μg/well) were isolated on 10% sodium dodecyl sulfate and polyacrylamide gels, and the obtained bands were electronically transferred onto polyvinylidene fluoride membranes, followed by blockage with Tris-buffered saline (TBS) and 5% skimmed milk at room temperature for 60 min. The membranes were incubated at 4°C with primary antibodies: anti-PTEN (1:1000, SAB1406331, Sigma), anti-phospho-Akt (1:500, SAB4504331, Sigma), anti-Akt (1:2000, SAB4500797, Sigma), and anti-Actin (1:5000, A2066, Sigma) antibodies. The membranes were washed twice with TBS, to which 0.1% Tween-20 was added prior to incubation with secondary antibodies. The Ag/Ab complex was examined using the enhanced chemiluminescence detection kit with β-actin as the loading control.

Cells were lysed with lysis buffer, and proteins were extracted using a protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. The proteins were separated by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electrotransferred onto polyvinylidene difluoride membranes. The membranes were blocked for 2 h with 5% non-fat milk at room temperature. Then, the membranes were incubated with primary antibodies against RBM8A (1 : 1000, HPA018403, Sigma-Aldrich), mTOR (1 : 1000, SAB4501039, Sigma-Aldrich), p-mTOR (1 : 1000, SAB4301415, Sigma-Aldrich), p-AKT (1 : 1000, SAB4504331, Sigma-Aldrich), AKT (1 : 1000, SAB4500797, Sigma-Aldrich), and β-actin (1 : 5000, SAB2701711, Sigma-Aldrich) at 4°C overnight. The membranes were then incubated with HRP conjugated secondary antibody (1 : 5000, 12–348, Sigma-Aldrich) for 1 h at room temperature. The immunoblots were measured by chemiluminescence detection system and quantified by ImageLab software (Bio-Rad, Hercules, CA, USA).