Western Blotting Protein Analysis

Western blotting was performed as described previously (6 (link)). Cells were lysed using lysis buffer and the concentration was analyzed by the BCA assay (Beyotime Biotechnology). Whole-cell lysates were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Shanghai, China), and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST). The primary antibodies used in the study included anti-AKT (1:1,000, Affinity Biosciences, Changzhou, China), anti-p-AKT (1:1,000, Cell Signaling Technology, Shanghai, China), anti-p38 MAP kinase (1:1,000, Affinity Biosciences), anti-phosphorylated p38 (1:1,000, Cell Signaling Technology), and anti-β-actin (1:2,000, Sangon, Shanghai, China). HRP-conjugated species-specific secondary antibodies were purchased from Beyotime Biotechnology. Signals were generated by enhanced chemiluminescence (Biosharp, Hefei, China) and captured by the Odyssey Fc system (LI-COR Biosciences, Lincoln, NE).