Ab191032

Total protein content was extracted with the help of a radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor and phosphorylase inhibitor, and the obtained protein concentration was analyzed using bicinchoninic acid kits (Boster, A53226, Thermo Fisher Scientific). After undergoing electrophoresis separation, the proteins were transferred onto a polyvinylidene fluoride membrane (IPVH85R, Merck Millipore, Billerica, MA). Following blockade with 5% bovine serum albumin (BSA), the membrane was probed with primary antibodies (Abcam Inc., Cambridge, UK) against HDAC8 (ab187139, 1 : 10000), IRF1 (ab191032, 1 : 1000), SUCNR1 (ab272856, 1 : 1000), LC3 (ab128025, 1 : 1000), and GAPDH (ab181602, 1 : 2500, normalizer) overnight at 4°C. The following day, the membrane was reprobed with the horseradish peroxidase- (HRP-) labeled secondary antibody IgG (ab6721, 1 : 5000, Abcam) for 2 h. Afterwards, the immunocomplexes were visualized with an enhanced chemiluminescence reagent, and band intensities were quantified using the ImageJ 1.48u software (V1.48, National Institutes of Health, Bethesda, Maryland).

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