Tissues were lysed with 1 mL RIPA lysis buffer and supplemented 1% protease inhibitor cocktail (Solarbio) for 2–5 min with steel balls in a tissue lyser (Qiagen, Germany). Tissue lysates were incubated with the buffer at 4 °C overnight (cell samples were incubated in lysis buffer for 30 min at 4 °C). The lysate was centrifuged at 12,000 rpm for 10 min, the supernatant was collected, and the protein concentration was determined using the BCA method (P0011, Beyotime Biotechnology). The supernatant was mixed with loading buffer and boiled at 100 °C for 10 min. Samples were loaded and subjected to 10% SDS-PAGE. After transfer to PVDF membranes, blots were blocked with 5% skimmed milk powder and incubated with primary antibody overnight at 4 °C. The primary antibodies included a BATF antibody (8638 S, Cell Signaling Technology, USA), anti-β-actin (GB11001, Servicebio, China), and β-tublin (15115, Cell Signaling Technology). Secondary antibody was goat anti-rabbit (Jackson, 111-035-003), images were acquired using the BIO-RAD protein gel imaging system (Universal hood II), and band intensity was calculated using Image J (Zhang et al., 2021c (link)).
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