0.45 μm polyvinylidene difluoride membranes

Cells and tissues were collected and lysed in cell/tissue lysis buffer (Solarbio, Beijing, China). Proteins were extracted, and the protein concentration was measured by the BCA method (Solarbio, Beijing, China). Proteins were separated by 10% sodium dodecyl sulfate (SDS)-PAGE and transferred to 0.45-μm polyvinylidene difluoride membranes (Invitrogen, CA, USA), which were blocked with blocking buffer (EpiZyme, Shanghai, China), incubated overnight with primary antibodies, and washed and incubated with the HRP-conjugated anti-rabbit secondary antibody (Abcam, UK) for 1 h. Peroxidase activity was visualized via the ECL method (EpiZyme, Shanghai, China).

The cell lysate was prepared with SDS-lysis buffer (Beyotime, Shanghai, China). Total protein concentration was measured by Nano-100 Micro-Spectrophotometer. Protein was then separated by 10% SDS-polyacrylamide microgel and transferred to 0.45-μm polyvinylidene difluoride membranes (Invitrogen). The PVDF membranes were then blocked in Tris-buffered saline containing 5% nonfat dry milk (w/v) (Sangon Biotech) in Tween-20 (TBST) for 1 h at room temperature. After that, the membranes were incubated with the indicated primary antibodies overnight. Then, they were incubated with a secondary antibody conjugated with HRP. We used Tanton™ Chemistar High-sig ECL Western Blotting Substrate (ECL) to display the signal. Full blots of images cropped for presentation are presented in Supplementary Material.