Ffp19

For protein from cells, cells were washed with cold PBS three times and subsequently 200 μl RIPR containing 2 μl protease inhibitors was added into 6 wells for 30 min at 4°C. For protein from tissues, fresh tissues were washed with cold PBS and RIPR was added to tissues in EP tubes. Then, EP tubes were moved to high throughput homogenization (Scientz, Ningbo, Zhejiang, China) for 1 min in 60 Hz. Lysates were centrifuged at 12000 rpm/min for 30 min at 4°C, and then the supernatant was moved to new Eppendorf tubes and stored at −20°C until used. 5 μg protein and 5x SDS-PAGE loading buffer (93-2108-10, MultiSciences, Hangzhou, Zhajiang, China) were added into each EP tubes to 1x, heated for 5 min at 95°C. Protein was separated on 10% SDS-PAGE (P1200, Solarbio, Beijing, China) gels and then transferred into PVDF membranes (FFP19, Beyotime, Shanghai, China). Membranes were washed with TBST for 15 minutes at room temperature. PVDF membranes were blocked with 10% milk for 1 hour at room temperature. After washed, PVDF was incubated with primary antibody overnight at 4°C in shaking table. Then, membranes were washed for 30 minutes with TBST and incubated appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Proteins were detected with Thermo Scientific Pierce ECL (32106, Thermo, Waltham, Massachusetts, USA) after washing three times for 10 minutes.