Anti histone h3

Protein samples were dissolved in SDS-PAGE loading buffer, boiled for 5 min, and loaded on 10% (v/v) polyacrylamide gel. After running the gel at 100 V for ~1 h, proteins were electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane using Semidry Trans-Blot apparatus (Bio-Rad) at 20 V for 20 min. The membrane was blocked in TBST buffer containing 5% (w/v) nonfat milk for 1 h, followed by washing three times with TBST buffer. The membrane was incubated for 1 h with primary antibodies: anti-Flag (GenScript A00187; 1:2000 diluted), anti-6xHis (GenScript A00186; 1:2000 diluted), anti-Histone H3 (GenScript A01502; 1:1000 diluted), anti-PEPC (Rockland 100-4163; 1:500 diluted), and anti-GAPC (Agrisera AS15-2894; 1:2000 diluted). After washing three times with TBST buffer, the membrane was incubated with secondary antibodies (Sigma-Aldrich) from mouse or rabbit conjugated with alkaline phosphatase for 1 h. Proteins were visualized by alkaline phosphatase conjugate substrate (Bio-Rad) according to the manufacturer’s instructions.