Protein Extraction and Western Blot Analysis

As previously described [14 (link)], total protein from each sample was extracted with RIPA lysis buffer (Sigma, St, Lousis) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF, Solarbio). A phosphorylase inhibitor and a protease inhibitor cocktail (1:100, Solarbio) were added to prevent degrading of proteins in the extracts. tThe protein concentration was quantified using a BCA Protein Assay Kit (Beyotime, China). Afterwards, the equal boiled protein samples (30 μg) were loaded on 8 ~ 12% sodium dodecyl sulfatepolyacrylamide gels (Solarbio Life Sciences), electrophoretically separated, and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Massachusetts), which had been wetted in 100% methanol for 15 s previously.. Then the membranes were blocked in 5% skim milk containing Tris-buffered Saline Tween (TBST, Solarbio, China) at room temperature for 1 h to reduce nonspecific bindings. Subsequently, the membranes were incubated with the primary antibodies (1:500 ~ 1:1000 dilutions, Cell Signaling Technology, USA) overnight at 4 °C with gentle agitation. Getting washed 2 ~ 3 times with TBST, the secondary antibodies (1:2000 dilution) were added for incubation for 1 h. Finally, the immunoreactive bands were detected with Chemiluminescent HRP Substrate (Merck Millipore) and quantified through a Image Lab software (Bio-Rad, Hercules, USA) .