XB-IMG-171813
Xenbase Image ID: 171813
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Figure 3. The Xenopus NuRD Complex Has DNA Replication Initiation ActivityNuRD was immunoprecipitated with MTA2-specific antibodies from the 20%–45% ammonium sulfate fraction of the activated Xenopus laevis egg extract.(A) Co-immunoprecipitation of GATAD2/p66 and HDAC1 subunits with MTA2, as confirmed by western blotting. Input (In), supernatant (Su), and washed pellet (P) of the control and MTA2 coIPs are shown, 10% of the total was loaded per lane for each.(B) Mass spectrometry analysis of co-immunoprecipitated material. Xenopus proteins (n = 447) uniquely identified only in the immunoprecipitate and not in the control IP were ranked according to their quantitative emPAI values after normalization to emPAI (MTA2) = 1. The top 14 most abundant proteins are plotted together with additional NuRD subunits. NuRD subunits are shown in blue. The mean normalized emPAI value of all proteins is indicated by a dashed line. Separate and additional isoforms were identified in the Uniprot database for GATAD2/p66 (∗LOC398154; ∗∗LOC100158394, isoform X2) and MTA2 (∗∗∗MGC83056), respectively.(C) Immunodepletion. xNuRD was co-immunoprecipitated with MTA2-specific antibodies as indicated above. Control immunoprecipitations were performed with either empty beads (mock) or with antibodies specific for Drosophila Vasa protein. The indicated protein amounts of immunodepleted supernatants were added to Y RNA-depleted DNA replication initiation reactions. Mean values ± SD of percentages of replicating template nuclei are plotted from n = 3 independent experiments. Brackets indicate results of t tests (unpaired, two-tailed with unequal variance) of experimental and control samples against the untreated input samples (ns, not significant; ∗∗∗p ≤ 0.001).See also Table S2. Image published in: Christov CP et al. (2018) © 2018 The Author(s). 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 |