Western Blot Protein Expression Analysis

Western blot was performed as previously described [16 (link)]. Briefly, cells were lysed on ice for 30 mins using cell lysis buffer (50 mM Tris HCl, pH 7.4, with 250 mM NaCl, 1 mM EDTA, 50 mM NaF, and 0.5% Triton X-100, together with protease inhibitors). Then the lysis was centrifugation at 12000 rpm for 15 mins at 4 °C. The protein concentration of each sample was determined using Bradford Kit (Sangon Biotech. Shanghai, China). For immunoblotting, proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Immunoblots were developed in Western Lightning Chemiluminescence Reagent Plus (Advansta, Menlo Park, CA, USA).