Figure 6

SMARCA5 associates with nascent DNA. A. Serum-starved NIH3T3 cells were released into S-phase in the presence of DMSO or 3 μM 898. Chromatin extracts were prepared at 0, 12 h, 18 h, and 24 h following release into S-phase for western analyses to look at SMARCA5 levels. B. Chromatin was prepared from either non-targeting (NT) siRNA or Hdac1,2 (H12) siRNA transfected cells at 72 h time-point to look at SMARCA5 levels. TATA binding protein (TBP) serves as a loading control. C. HeLa cells were labeled with bromodeoxyuridine (BrdU) for 30 min. Cells were then washed to remove unincorporated BrdU and cultured in media without BrdU for indicated periods of time (chase). Chromatin immunoprecipitation (ChIP) was performed with anti-proliferating cell nuclear antigen (PCNA) antibody. BrdU labeled DNA present in input DNA and those associated with PCNA were assessed in slot blot analysis using an anti-BrdU antibody. D-E. BrdU-pulse chase coupled to slot blot analysis in HeLa (D) or NIH3T3 (E) cells to determine the kinetics of SMARCA5 association to nascent DNA at the indicated chase time points. F-G. NIH3T3 cells were treated with either DMSO or 898 for 24 h or transfected with non-targeting or Hdac1,2 siRNA and labeled with 20 μM BrdU for 1 h before harvesting. ChIP of SMARCA5 or rabbit IgG control was done and increasing volumes of ChIP DNA were spotted onto a membrane using slot blot and probed with anti-BrdU antibody. In F-G, a representative blot from three independent experiments is shown. UT refers to untreated.