Figure 3. Usp7 binds to replicating chromatin in a Dnmt1-dependent manner. (A) CSF extracts (M-phase) were released into interphase by addition of CaCl2. Sperm nuclei were added to either CSF or interphase extracts. Aphidicolin (APD, 150 μM) was added to extracts 10 min before sperm addition. At the indicated times after incubation, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. (B) Sperm chromatin was added to mock- or xDnmt1-depleted interphase extracts. xDnmt1-depleted extracts were supplemented with either buffer alone (lanes 9–12), purified wild-type xDnmt1-FLAGx3 or its 4KA mutant-FLAGx3 (10 ng/μl final concentration, lanes 13–16 or 17–20, respectively). At the indicated time points, chromatin fractions as well as extracts were subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. (C) Sperm chromatin was added to mock-depleted or xUhrf1-depleted interphase extracts. xUhrf1-depleted extracts were supplemented with either buffer alone (lanes 7–9) or purified hUhrf1 (30 ng/μl final concentration, lanes 10–12). At the indicated time points, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. Note that xUhrf1 antibodies recognized hUhrf1. ∆ Denotes immunodepletion of the protein indicated. (D) Either mock- or Dnmt1-depleted HSS were incubated with chromatin fractions from mock- (M) or Dnmt1- (D) depleted extracts. The resultant mixtures were immunoprecipitated with anti-histone H3 antibodies and subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. Source data are available online for this figure.
Image published in: Yamaguchi L et al. (2017)
© The Author(s) 2017. 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
Permanent Image Page
Printer Friendly View
XB-IMG-157221