Anti xpc

Briefly, cells were washed twice with ice-cold PBS and lysed in lysis buffer containing 50 mM Tris pH 7.4; 1% Triton X-100; 0.5 mM EDTA; 0.5 mM EGTA; 150 mM NaCl; 10% Glycerol; 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail (Roche) on ice for 30 min. The supernatant was collected after centrifugation at 15,000 × g for 15 min at 4 °C, and 500 µg of total cell lysate was treated by DNase (15 U mL⁻¹, Pierce), precleared by 20 µL Protein G Agarose Beads (Thermo Fisher Scientific Inc.), and then incubated with primary antibody, including anti-DOT1L (ab72454, Abcam) and anti-XPC (#14768, Cell signaling) overnight at 4 °C. In all, 20 µl of Protein A/G Agarose Beads was added into the samples with rotation at 4 °C for 1 h. After three washes with 1 mL of lysis buffer, the bound proteins were released by boiling in 30 μL of sodium dodecyl sulfate loading buffer and detected as described above. The chromatin fractionation was extracted with the Chromatin Extraction Kit (ab117152, Abcam) from the cells and assayed with western blot.