Anti ddup antibody

The indicated cells (1 × 10⁶) were seeded in 10 cm dish and cultured overnight. Then cells were harvested after treatment with CPT or CDDP and resuspended in 200 μl of buffer A (10 mM KCl, 15 mM MgCl₂, 10 mM HEPES, [pH 7.9], 0.34 mM sucrose, 1 mM dithiothreitol, 10% glycerol, 0.1% Triton X-100 and protease inhibitor mixture), and incubated for 10 min on ice. The cell pellet was collected at 1300 g for 4 min at 4°C. After being washed with buffer A, the cell pellet was lysed in 200 μl of lysis buffer (0.2 mM EGTA, 1 mM dithiothreitol, and protease inhibitor mixture). Insoluble chromatin was harvested by centrifugation (1700 g, 4 min, 4°C).
The chromatin fraction was then treated with DNAase (stem cell Technologies, CA) for 1 h and then incubated with anti-DDUP antibody (Sino Biological, China) overnight at 4°C. After then the supernatant was incubated with 20 μl of protein G-agarose beads overnight at 4°C. The agarose beads were washed six times with wash buffer (25 mM HEPES [pH 7.4], 0.5% NP-40, 1 mM EDTA, 150 mM NaCl, 2% glycerol, 1 mM PMSF). The precipitated components were subjected to LC–MS/MS or western blot analysis. The protein level in LC–MS/MS analysis were quantified by following thresholds, which were considered to be significantly differentially abundant: pFDR value <0.05, fold change ≥2.

Immunoblotting was performed by using the proteins immunoprecipitated by anti-ATR (ab2905, Abcam), anti-RAD18 (ab188235, Abcam), anti-human gamma H2A.X (phosphor S139) (ab81299, Abcam) and anti-RAD51C (A302-645A, Bethyl Laboratories) antibodies. Briefly, the indicated cell lysates were separated by SDS-PAGE, and were transferred onto a PVDF membrane. The membrane was then preincubated in 10% skimmed milk at 4°C for 1 h. Then, the human recombinant DDUP protein (Abclonal, China) was added and further incubated for 18 h at 4°C. The membrane was then washed six times with TBST buffer and subjected to immunoblotting analysis by anti-DDUP antibody (Sino Biological, China).