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. Author manuscript; available in PMC: 2018 Oct 5.
Published in final edited form as: Mol Cell. 2017 Sep 21;68(1):61–75.e5. doi: 10.1016/j.molcel.2017.08.021

Figure 6. ASF1a is required for RNF8, but not MDC1, recruitment at DSBs.

Figure 6

(A to C) Decrease of bleomycin-induced RNF8 foci, but not MDC1 foci in ASF1a knockout cells. HeLa DR13-9 wild type or ASF1a -/- cells were incubated with 20 μgml-1 bleomycin for 40 min followed by fixation. Representative images of immunostaining with indicated antibodies (A and B), and quantification of cells with >5 foci of RNF8 or MDC1 (C). See colocalization of RNF8 and MDC1 foci in Figure S3. Scale bar, 10 μm. Mean ± S.D. of triplicates. ***, P < 0.005. (D) Decreased interaction of MDC1 with RNF8 after DNA damage in ASF1a knockout cells. Plasmid expressing Flag-RNF8 was transfected in 293B wild type and ASF1a -/- cells followed by bleomycin treatment before harvest. Asterisk indicates immunoglobulin heavy chain. (E to I) ASF1a is required for RNF8/168 localization, not MDC1, at I-SceI cut site. HeLa DR13-9 cells transfected with indicated siRNAs (E to G), wild type and ASF1a -/- cells (H and I) were applied to ChIP assay using anti-RNF8 (E and H), -MDC1 (F and I) and -RNF168 (G) antibodies. Mean ± SD of triplicates. ***, P < 0.005; *, P< 0.05.