Histone h3

RAW264.7 cells were treated with RANKL or carnosol (1 µM) in 6-well plates for 0, 5, 10, 15, 30 and 60 min. The protein was extracted by RIPA Lysis Buffer (cat. no. BL504A; Biosharp) and assessed by the BCA protein assay kit (cat. no. BL521A; Biosharp). 100 µg protein was loaded per lane. The percentage of gel for Histone H3 was 15% and for others were 12%. PVDF membrane was used for transfer. The blots were probed with primary monoclonal antibodies against mouse TRAP (1:2,000; cat. no. ab191406; Abcam), cathepsin K (1:2,000; cat. no. ab187647; Abcam), MMP-9 (1:2,000; cat. no. ab76003; Abcam), p65 (1:2,000; cat. no. 10745-1; ProteinTech Group, Inc.), phosphorylated (p)-p65 (1:2,000; cat. no. ab76302; Abcam), IκBa (1:5,000; cat. no. ab32518; Abcam), β-actin (1:2,000; cat. no. GB11001; Servicebio, Inc.) and Histone H3 (1:1,000; cat. no. ab176842; Abcam). Following primary incubation (4°C, overnight), membranes were washed by TBST (0.1% Tween-20) and incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody for 1 h at 4°C (1:10,000; cat. no. ab205718; Abcam). After washing, the bands were detected via ECL Western Blotting substrate (cat. no. BL520A, Biosharp) and were imaged using the Bio-Rad ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.).