Western Blot Analysis Protocol

Western blot analyses were carried out as described previously [4 (link), 28 (link)]. Briefly, LPCs were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013D, Beyotime) supplemented with protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (PhoSSTOP Cocktail Tablets, Roche) on ice, after which the concentration of the proteins was measured via BCA protein assay kits (Beyotime). Equal amounts of cell lysate were then mixed with sodium dodecyl sulfate (SDS) loading buffer, denatured, loaded and separated via SDS–polyacrylamide (SDS–PAGE) gel (Bio-Rad, Bio-Rad, Hercules, CA, USA). After separation, the proteins were transferred to PVDF membranes (Roche), after which the proteins were incubated with primary antibodies overnight at 4°C. After incubation, the membrane was washed with TBST 3 times (5 min each) and then incubated for 1 h with a horseradish peroxidase (HRP)-labeled secondary antibody at 37°C. Finally, the membrane was washed again in TBST 3 times, reactive bands on the membrane were detected by enhanced chemiluminescence (ECL) reagent (Pierce Protein Biology, Thermo Fisher Scientific, Waltham, MA, USA), and the signals were visualized by an image labeling imaging system (Bio-Rad).