Rabbit anti akt antibody

The cells were collected and lysed in Western and IP lysis buffer containing PMSF for 5 min on ice, followed by centrifugation at 13,000 × g for 25 min at 4°C. The supernatant was harvested, and the protein concentration was quantified using a BCA protein assay kit. Western blot analysis was carried out by standard protocol. The following antibodies were used: rabbit anti-TIMMDC1 antibody (1:1000), rabbit anti-Phospho-AKT antibody (1:1000), rabbit anti-Phospho-GSK-3β antibody (1:1000), rabbit anti-β-catenin antibody (1:1000), rabbit anti-AKT antibody (1:1000), rabbit anti-GSK-3β (1:1000), rabbit anti-c-Myc antibody (1:1000) were from Abcam Inc. Mouse anti-β-actin antibody (1:1000, AA128), HRP-labeled goat anti-rabbit IgG (1:500, A0208), and HRP-labeled goat anti-mouse IgG (1:500, A0216) were from Beyotime Institute of Biotechnology (Nanjing, China).

Total protein was separated in 10% sodium dodecylsulfate polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck, Darmstadt, Germany). The membranes were blocked in Tris-buffered saline Tween 20 (TBST) solution containing 5% skim milk at room temperature for 2 h, and then probed overnight with primary antibodies at 4°C. The following antibodies were used: Rabbit anti-Akt antibody (1: 2000, Abcam, UK), rabbit anti-p-Akt antibody (1: 2000, Abcam, UK), rabbit anti-Bad antibody (1: 1000, Abcam, UK), rabbit anti-p-Bad (1: 1000, Abcam, UK), and β-actin (Sigma, USA). The membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 1000, Abcam, UK) at room temperature for 1 h.