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Fig. 6. ChIP assay for in vivo interaction between PARP and T-T. (A) Co-immunoprecipitation of DNA containing T-T photolesions with PARP. ChIP input DNA (top two panels): Chromatin extracts of control or UVB (1.6 and 6.4 kJ/m2 for 15 seconds)-irradiated cells were adjusted prior to ChIP to 100 µg input DNA and an equal aliquot from each extract was assayed for DNA content by ethidium bromide staining and for T-T content by immunodot-blot. ChIP eluate using antibodies to PARP (bottom two panels). DNA was extracted from PARP-ChIP eluates of control and UVB-treated cells, and an equal portion of each eluate was assayed for DNA content by ethidium bromide and for T-T content by immunodot-blot. (B) Recovery of PARP cross-linked to T-T. ChIP was carried out for extracts from control and UVB (6.4 kJ/m2)-irradiated cells with anti-T-T, and proteins eluted in SDS-PAGE sample buffer were immunoblotted for PARP. The 25 and 50 kDa bands are subunits of antibodies used in immunoprecipitation. Apoptotic HL-60 cells were loaded for identification of PARP. Data are from one of five experiments with identical results.
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