Anti myc

DNAP assay was performed as previously described [23] (link). The biotin-labeled DNA probes (Sigma-Aldrich) were annealed to complementary oligonucleotides. COS-7 cells were transfected with 1 µg of expression vectors using LipofectAmine 2000 (Invitrogen), according to the manufacturer's instructions. Transfected COS-7 cells or nuclear fractions of BM-DCs were lysed or diluted with DNAP binding buffer [25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.25% NP-40, 1 mM DTT and Complete Protease Inhibitor Cocktail (Nacalai Tesque)], respectively. Cell debris was removed by centrifugation (20,000×g) for 10 min. Lysates were first incubated with Streptavidin-Sepharose beads (GE Healthcare) for 30 min to eliminate nonspecific binding and then incubated with 1.5 µg of poly(dI-dC) and 2 µg of biotinylated DNA probe for 1 h at 4°C. Streptavidin-Sepharose beads were then added and incubated with these mixtures for an additional 30 min at 4°C. After washing the beads three times in DNAP binding buffer, precipitated proteins were eluted in SDS-PAGE sample buffer. Samples were analyzed SDS-PAGE followed by Western blot analysis using anti-Sp1 (Santa Cruz Biotechnology), anti-Myc (Nacalai Tesque), anti-RARα (Santa Cruz Biotechnology), and anti-RXRα (Santa Cruz Biotechnology) Abs.

Anti-RAD51 (1:100 Santa Cruz, sc8364), anti-DMC1 (1:50 Santa Cruz, sc22768), anti-SYCP3 (1:500 abcam, ab97672), anti-γH2AX (1:500 Millipore, 05–636) antibodies were used for immunofluorescence staining of spermatocyte spread. Anti-RAD51 (1:500 Millipore, ABE257) and anti-γH2AX (1:1000 Millipore, 05–636) antibodies were used for immunofluorescence staining of U2OS cells. Anti-FLAG (1:3000 Wako 012-22384) and anti-Myc (1:3000 Nacalai 04362-34) antibodies were purchased from Wako and Nacalai, respectively. For pulldown assay, anti-FIGNL1 (1:500 abcam, ab173685) and anit-SWSAP1 (1:500 Thermo, PA5-25460) were used.