Western Blot Analysis Protocol

WB analysis was performed as described previously [28 (link),29 (link)]. Briefly, cells were lysed with WB/IP lysis buffer (Beyotime Biotechnology, P0013, Shanghai, China) containing protease inhibitor cocktail (Roche, 11697498001, Germany). Cell extracts were subjected to 10% SDS-PAGE, transferred onto 0.45 μm PVDF membranes (Millipore, Darmstadt, Germany), and blocked with 5% non-fat milk at 4 °C overnight. The membrane was clipped and probed with a primary antibody at room temperature for 2 h. After washes with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (BOSTER, BA1054, Wuhan, China) or goat anti-mouse IgG (BOSTER, BA1051, Wuhan, China) at room temperature for 1 h. Protein bands were exposed in an Image Lab^TM System (BIO-RAD, Contra Costa County, CA, USA) after the addition of chemiluminescent substrate (Advansta, K-12045-D50, San Jose, CA, USA). The relative intensities of Western blots were quantified using Image J. A protein molecular weight marker was purchased from Bioscience (Double Helix, P12083, Shanghai, China).