Gpr109a

The tissues were lysed in lysis buffer (Beyotime, Shanghai, China). The protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA). A total of 30 μg protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis SDS-PAGE. The separated proteins were subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany). After blocking with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against histone H3 (1:2000) (Cell Signaling Technology, Danvers, MA, USA), citrulline histone H3 (1:1000) (abcam, UK), GPR109A (1:500) (Santa Cruz Biotechnology, California, USA) and β-actin (1:8000) (proteintech, Wuhan, China). Subsequently, the membrane was washed five times in 0.05% Tris-buffered saline with Tween-20 (TBST, pH 7.4) for 10 min each time and was then incubated with an HRP-conjugated anti-mouse (1:20,000) or anti-rabbit secondary antibody (1:10,000) (Bosterbio, USA) for 1 h on a shaker at RT. The membrane was again washed five times for 10 min each with TBST. Finally, a SuperEnhanced chemiluminescence detection kit (Applygen Technologies Inc, Beijing, China) was used to visualize the immunoreactive proteins. The protein bands were visualized after exposure of the membranes to X-ray film.