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Figure 4. Human NuRD Complexes Are Structurally and Functionally Distinct from xNuRD(A–F) Proliferating human HeLa cells were fractionated into nuclear (A–C) and cytosolic extracts (D–F), and each extract was partially sub-fractionated by precipitation with 20%–45% ammonium sulfate and ultracentrifugation through preparative sucrose gradients.(A and D) Western blot analyses of human NuRD subunits. Fractions of the preparative sucrose gradients of nuclear (A) and cytosolic extracts (D) were analyzed by western blotting with antibodies specific for the indicated human NuRD subunits.(B and E) Mass spectrometry analysis of the MTA2 co-immunoprecipitations. Fractions 6–8 of the preparative sucrose gradients of nuclear (B) and cytosolic extracts (E) were pooled, and human NuRD was immunoprecipitated with anti-MTA2 antibodies. Human proteins identified in the immunoprecipitates (n = 126 and n = 183 for nuclear and cytosolic extracts, respectively) were ranked according to their quantitative emPAI values after normalization to emPAI (MTA2) = 1, and all human NuRD subunits detected are plotted with their rank. The mean normalized emPAI values of all proteins are indicated by dashed lines.(C and F) Activity profiles of the sucrose fractions. Template nuclei were incubated in Y RNA-depleted human cytosolic extract supplemented with the indicated gradient fractions of the nuclear (C) and cytosolic extracts (F). Mean values ± SD of percentages of replicating template nuclei are plotted of n independent experiments. Brackets indicate results of t tests (unpaired, two-tailed with unequal variance) of the positive control (xNuRD) and experimental samples against the Y RNA-depleted background (∗∗∗p ≤ 0.001).See also Figures S2 and S3 and Table S3. 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

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