Enhanced chemiluminescent plus substrate

The whole cells were washed and lysed in RIPA buffer (89900, Thermo Scientific) supplemented with PMSF (phenylmethylsulfonyl fluoride) and protease inhibitors. Protein concentrations were determined with a BCA assay kit (Pierce, Rockford, IL, USA). Then the lysates were mixed with loading buffer, analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocked with 5% skim milk in TBST at room temperature for 1 h, the membranes were incubated with different primary antibodies, including anti-DDX54 (1:1000, Proteintech, China), anti-P65, anti-p-P65, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, (1:1000, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (1:5000, Yeason, China) in 5% milk/TBST buffer at 4°C overnight, and then probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h. After washing three times with TBST, the membrane was developed with enhanced chemiluminescent plus substrate (Merck Millipore, Billerica, MA, USA), and the signal was recorded by Fluorchem E System (ProteinSimple, Santa Clara, CA, USA).

Whole cell lysates were prepared in RIPA buffer (Thermo Scientific) with phenylmethylsulfonyl fluoride and protease inhibitors and centrifuged. Supernatants were aliquoted, mixed with loading buffer, resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidenedifluoride membranes (Millipore, Billerica, MA). After blocked with 5% skim milk in TBST (Tris-buffered Saline with Tween 20) at room temperature for 1 h, the membranes were incubated with different primary antibodies, including anti-ERK, anti-p-ERK, anti-P65, anti-p-P65, anti-cyclin D1 (1:1000,Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (1:5000, Yeason, China) in 5% milk/TBST buffer at 4 °C overnight, and then probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h. After washing with TBST, the membrane was developed with enhanced chemiluminescent plus substrate (Merck Millipore, Billerica, MA, USA) and the signal was recorded by Fluorchem E System (Protein Simple, Santa Clara, CA, USA).