Ab229136

A total of 2 × 10⁶ cells were used to extract protein samples with a mixture of 100μlRIPA buffer (Beyotime, # P0013B) and 2 μl PMSF (Sigma, #329-98-6). The concentrations of protein were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, #NCI3225CH). The total protein (50 μg/sample) added in the loading buffer was boiled at 100 °C for 5 min, separated in a 10% gel at 80 V for 90 min, transferred to a membrane at 4°C using 300 mA for 85 min, and then incubated with QuickBlock™ Western (Beyotime, #P0252) for 10min. The membranes were probed with antihuman TET1 antibody (Abcam #ab191698), anti-OASL (Abcam #ab229136), anti-IRF1 (Abcam #ab191032), and anti-β-actin (Abcam #ab8227)) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Abcam#) served as a secondary antibody

Part of the tissues of TT and TP in PA patients was homogenized in lysis buffer containing protease and phosphatase inhibitor (Sigma-Aldrich). Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose membrane. After blocking, incubate with TRPM2, GAPDH, Ras, Raf-1, PSPH, OASL, PKC, METTL3, HIST1H2AE, cPLA, and AQR antibodies (abcam, ab11168, ab9482, ab52939, ab173539, ab211418, ab229136, ab205791, ab195352, ab18255, ab53421, ab205303) at 4 °C overnight. After incubating with the secondary antibody at 20 °C for 1 h, the membrane was washed 3 times. The immune response zone is detected by the ECL detection system. The protein bands were quantified using Image J software (NIH, Bethesda, MD, USA).