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. 2017 Nov 6;8:1333. doi: 10.1038/s41467-017-01080-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — DDB2-dependent recruitment of ASH1L to chromatin. a Recruitment of DDB2, XPC and ASH1L to chromatin detected by immunoblotting. The chromatin of HeLa cells exposed to UV-C (10 J m^–2) was analyzed, at different times, by salt extraction to release free proteins, followed by MNase solubilization. GAPDH and H3 were loading controls for free proteins and solubilized chromatin, respectively. Ub, ubiquitinated XPC protein. b Quantification of ASH1L recruitment to chromatin normalized to H3 (n = 4 independent experiments). ASH1L levels in unirradiated chromatin are set to 100%; error bars show s.e.m. and significance was calculated using the one-sample t-test. c Increased H3K4me3 levels following UV radiation (10 J m^–2). Whole cell lysates were probed with antibodies against the indicated forms of H3. Numbers at the bottom of each blot indicate the quantity of methylated H3 normalized to total H3 and relative to unirradiated controls. d Quantification of H3K4me3 and H3K36me3 normalized to total H3 (n = 6 independent experiments, one-sample t-test), values in unirradiated controls are set to 100%. e DDB2-dependent recruitment of ASH1L to chromatin. HeLa cells were siRNA-transfected as indicated 48 h before UV radiation (10 J m^–2). After a 1-h recovery, cells were analyzed by salt extraction, chromatin solubilization and immunoblotting. NC, non-coding. Numbers at the bottom of the blot indicate ASH1L protein levels normalized to H3 and relative to the unirradiated control. f Quantification of ASH1L recruitment to chromatin, relative to free ASH1L, normalized to H3 and GAPDH (n = 4 independent experiments, one-sample t-test). g Co-immunoprecipitation (Co-IP) of ASH1L with DDB2. HeLa cells were siRNA-transfected 48 h before collection of 0.3-M NaCl extracts. These extracts were immunoprecipitated with anti-DDB2 antibodies and analyzed by immunoblotting; –Ig, immunoglobulins omitted (the DDB2-proficient extract was subjected to the same Co-IP procedure except that anti-DDB2 antibodies were left out). h Chromatin immunoprecipitation (ChIP) using antibodies against DDB2 demonstrating that DDB2 and ASH1L interact in the chromatin of UV-irradiated (10 J m^–2) HeLa cells; –Ig, immunoglobulins omitted (samples were subjected to the same ChIP procedure except that anti-DDB2 antibodies were left out)