Western blotting

Cell samples were lysed in the RIPA lysis buffer (Beyotimem, Shanghai, China) on the ice with 1% PMSF (Servicebio, Wuhan, China) and phosphatase inhibitor (Servicebio, Shanghai, China). Next, the lysates were centrifuged right away at 13,000 rpm for 15 min after being incubated on ice for 30 min. The supernatant from each sample was then collected, and the BCA assay was used to detect the concentration of sample protein. Then a 25% volume 5X Native Gel Sample Loading Buffer (Biosharp, Beijing, China) was added into the samples and boiled for 15 min at 100 °C. All protein samples were preserved at − 80 °C or − 20 °C for different types of storage. Equivalent protein from each sample was separated by 10% SDS-PAGE for 2 h at 80 V, and then protein was transferred to PVDF membranes (Millipore, NJ, USA) at a current of 200 mA. The membranes were then blocked with a protein-free rapid blocking buffer (Epizyme, Shanghai, China) for 15 min at room temperature. The membranes were then incubated with various primary antibodies overnight at 4 °C, followed by incubation with goat anti-mouse IgG (SA00001-1, Proteintech) or goat anti-rabbit IgG (SA00001-2, Proteintech) at room temperature for 1 h. Specific bands were tested with an ECL kit (Beyotime, Shanghai, China). Finally, a ChemiDoc™ Touch Imaging System (Bio-Rad) was used to scan protein bands and acquire images, and ImageJ software was used to analyze the results.