Ab237510

The harvested cells after the different treatments were lysed in RIPA buffer (Beyotime Biotechnology) containing 10 µM PMSF on ice for 20 min. After centrifugation at 4 °C for 14,000 g × 10 min, the supernatant was transferred into a new sterile tube. Subsequently, protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 20 µg protein was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Burlington, USA). After blocking with 5% non-fat milk, the membranes were incubated with the individual primary antibodies Rabbit anti-USP33 (ab237510, Abcam, MA, USA), rabbit anti-GAPDH (ab9485, Abcam), mouse anti-Bax (ab3191, Abcam), rabbit anti-Bcl-2 (ab182858, Abcam), rabbit anti-JAK2 (ab108596, Abcam), rabbit anti-STAT3 (ab68153, Abcam) and rabbit-STAT3 (phospho Y705) (ab267373, Abcam) at 4 °C overnight. After three washes by 1 × PBST, the membranes were incubated with HRP- conjugated rabbit anti-Goat IgG (31,403, Thermo Fisher Scientific) or Donkey anti-Mouse IgG (H + L) (A16011, Thermo Fisher Scientific) for 1 h at room temperature. Subsequently, the membranes were visualized by Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, MA, USA). The membranes were imaged by Image J software.