XB-IMG-128054
Xenbase Image ID: 128054
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Figure 7. Depletion of KDM2B causes a reduction in H2AK119ub1.(A) KDM2B knockdown results in a global loss of H2AK119ub1, as demonstrated by western blot for H2AK119ub1 in KDM2B knockdown and control cells. Western blot for total H2A is shown as a loading control. (B) Quantification of H2AK119ub1 levels by fluorescence based quantitative western blotting. The levels of H2AK119ub1 are approximately 40% lower in KDM2B knockdown compared to control cells. The western blot signal for H2AK119ub1 was quantified relative to H2A and the error bars represent the SEM of six biological replicates. (C) KDM2B knockdown does not cause global changes in levels of H3K27me3, H3K4me3 or H3K36me2, as demonstrated by western blot in the KDM2B knockdown and control cells. Western for total Histone H3 is shown as a loading control. (D) KDM2B knockdown cells show locus specific depletion of H2AK119ub1 at genes up-regulated in the KDM2B knockdown cell line. Comparatively there are only small changes in H3K27me3 and H3K4me3 at these same sites. ChIP material was analysed by qPCR using primers specific for (i) non-CGI promoters, (ii) gene bodies, (iii) Non-PcG target CGIs, (iv) PcG target CGIs, and (v) PcG target CGIs of genes up-regulated in KDM2B knockdown cells. Error bars represent the SEM of four biological replicates.DOI:
http://dx.doi.org/10.7554/eLife.00205.015Figure 7—figure supplement 1. Purification of an H2AK119ub1 antibody.A rabbit was immunized with a synthetic branched peptide containing H2AK119ub1. Serum from the immunized animal was tested against histone extracts from cells that contain a conditional allele of Ring1b that can be deleted by the addition of the drug tamoxifen. Drug treatment removes the RING1B H2AK119 E3 ligase and eliminates H2AK119ub1. In the crude serum from the immunized animal there is a clear H2AK119ub1 signal in the RING1B containing cell line that is lost upon drug treatment (panel i), however there was some cross-reactivity with unmodified H2A. To remove the H2A cross-reactivity the serum was first depleted of H2A reactivity (panel ii) and the flowthrough (panel iii) was then affinity purified on a column containing the H2AK119ub1 antigen (panel iv) yielding a highly specific antibody against H2AK119ub1 when compared to the commercially available H2AK119ub1 monoclonal antibody (panel v).DOI:
http://dx.doi.org/10.7554/eLife.00205.016 Image published in: Farcas AM et al. (2012) Copyright © 2012, Farcas et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |