Western primary antibody diluent

Western blot was performed as previously described:^{51 (link)} total protein was extracted from cell or tissue samples using RIPA buffer composed of 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate and complete protease cocktail (Roche) on ice. Samples were sonicated four times for 5 s on ice water at 15% power (Ningbo Scientific Biotechnology). Tissue lysates were centrifuged at 4 °C for 10 min at 14,000 g and supernatant was mixed with 4× LDS Sample Buffer (NP0007) and boiled for 10 min at 70 °C. The 10-μg protein samples were separated by 4–12% SDS–PAGE with MOPS-SDS running buffer and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were blocked with 5 QuickBlock™ Western Blocking Solution (P0252, Beyotime Biotechnology) for 1 h and then incubated with primary antibodies prepared using Western Primary Antibody Diluent (P0256, Beyotime Biotechnology) at 4 °C overnight. Membranes were incubated with HRP-conjugated secondary antibodies prepared using QuickBlock™ Western Secondary Antibody Diluent (P0258, Beyotime Biotechnology), and incubated for 1 h at room temperature. Immunoreactions were captured by X-ray films detecting chemiluminescence using an ECL kit (GE Healthcare). The full scan blots can be found in the Fig. S16.