Ab108928

To prepare extracts, tissues were collected from male C57BL/6 mice and suspended in lysis buffer [50 mM HEPES-KOH (pH 7.5), 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 10 mM NaF, 0.25 mM Na3VO4, and 50 mM β-glycerolphosphate] supplemented with cOmplete Protease Inhibitor (Roche #04693116001). After homogenization, the cell extracts were centrifuged at 13,000× g for 20 min at 4°C. The supernatant extracts were used for immunoprecipitation and Western blots. Equal amounts of protein were electrophoresed on 10% SDS–polyacrylamide gels, and the bands were transferred to polyvinylidene fluoride membranes (Millipore, USA). Immunoreactive bands were detected and analyzed with a Bio-Rad ChemiDoc MP imaging System and Image Lab Software (Bio-Rad, USA). Relative protein levels in each sample were normalized to β-Actin to standardize the loading variations. The primary antibodies for immunoblotting included anti-β-Actin (1:10,000 dilution; PTG, # 66009-1-Ig), anti-Lamin-B1 (1:3000 dilution; PTG # 12987-1-AP), anti-UAF1 (1:1000 dilution; PTG # 16503-1-AP), anti-USP1 (1:1000 dilution; PTG # 14346-1-AP), anti-KDM3A (1:1000, PTG # 12835-1-AP), anti-BRDT (1:1000, GeneTex # GTX100201), anti-MORC3 (1:1000 dilution; PTG #24994-1-AP), anti-FANCD2 (1:1000 dilution; Abcam # ab108928), and anti-PCNA (1:1000 dilution; Santa Cruz # SC56).