Enhanced chemiluminescence visualization kit

Cells were plated in 6-well tissue culture plates at 80% confluence and incubated overnight. Cell lysates were obtained from transduced cells using cold radioimmunoprecipitation assay buffer [20 mmol/l Tris-HCl (pH 8.0), 100 mmol/l NaCl, 10% glycerol, 1% NP40, 0.5% sodium deoxycholate]. Twenty micrograms of protein mixture were separated on 10% SDS-PAGE gels and wet transferred to nitrocellulose membrane (GE Healthcare Life Sciences) and then blocked for 1 h at room temperature in TBS-T buffer [50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 0.1% Tween-20] containing 5% non-fat milk. Membranes were then incubated overnight at 4°C or 1 h at room temperature with the respective primary antibodies: anti-CIP2A (1:500), and anti-actin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphor-mTOR (1:1,000), mTOR (1:1,000), phospho-AKT (S473) (1:1,000), (Cell Signaling Technology, Danvers, MA, USA). Anti-mouse or anti-rabbit secondary antibody-conjugated to horseradish peroxidase was used to visualize the stained bands with an enhanced chemiluminescence visualization kit (both from Santa Cruz Biotechnology).

Cells were plated in 6-well tissue culture plates at 80 % confluence and incubated overnight. Cell lysates were obtained from transduced cells using cold radioimmunoprecipitation assay buffer [20 mmol/L Tris–HCl (pH 8.0), 100 mmol/L NaCl, 10 % glycerol, 1 % NP40, 0.5 % sodium deoxycholate]. Twenty micrograms of protein were separated in 10 % SDS-PAGE gels and wet transferred to nitrocellulous membrane (GE Healthcare Life Sciences), then blocked for 1 h at room temperature in TBS-T [50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 0.1 % Tween 20] buffer containing 5 % nonfat milk. Membranes were then incubated overnight at 4 °C or 1 h at room temperature with the respective primary antibodies: CIP2A (1:500), pJNK (1:1,000), pATF2 (1:1000), phospho-c-Jun (1:1000) and actin (1:1,000). Anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used to visualize the stained bands with an enhanced chemiluminescence visualization kit (Santa Cruz Biotechnology).