Anti akt2

Cell protein lysates were separated in 8% or 10% SDS-PAGE gel 72 h post-transfection, followed by transferring to polyvinylidene difluoride membrane (PVDF). Western blot analysis was performed with monoclonal anti-p53 (Santa Cruz), anti-AKT2 (Abcam) primary antibodies. Anti-GAPDH antibody (Santa Cruz) was used as an internal control. The membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, USA). Complexes were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and the expression levels of these proteins were evaluated by Quantity One software.

Fifty micrograms of total protein from each sample was separated by 12% SDS/PAGE and electro-transferred onto PVDF membranes (Millipore, Boston, MA) for immunoblotting analysis. The primary antibodies including Anti-IGF2 (1:500, Abcam), Anti-AKT2 (1:500, Abcam), Anti-SHMT2 (1:300, Abcam), and Anti-β-actin (1:800, Abcam) were used to incubated with the membranes at 4°C overnight. After incubation with appropriate Horseradish Peroxidase-conjugated secondary antibodies, the blots were detected using Immobilon ECL Kit (Millipore) in a ChemiDoc XRS Imaging System and analyzed by Quantity One software (Bio-Rad).

Total proteins of cell samples were extracted with lysis buffer and then quantified using the BCA method (KeyGen Biotech, Jiangsu, China). The lysate was diluted in SDS sample buffer (KeyGen Biotech) for SDS-polyacrylamide gelelectrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche Applied Sciences,USA). The membranes were immunoblotted at 4 °C overnight with anti-PRDX2 (Proteintech, USA), anti-c-Myc(Abcam, UK), anti-p-c-Myc(S62) (Abcam), anti-p-c-Myc(T58)(Abcam), anti-E-cadherin (Proteintech), anti-Vimentin (Proteintech), anti-N-cadherin (Proteintech), anti-GSK3β (Abcam), anti-p-GSK3β (Ser9) (Abcam), anti-AKT(1/2) (Abcam), anti-AKT1 (Abcam), anti-AKT2 (Abcam), anti-p-AKT2 (Ser474) (Abcam) and anti-GAPDH (Proteintech) antibodies at appropriate dilution concentration, followed by incubation using the appropriate second antibodies for 2 h. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Image J software was used to analyze the grey value of the interest protein.