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. 2018 Feb 19;46(6):3218–3231. doi: 10.1093/nar/gky104

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — Both UHRF1 UBL and RING finger are critical for DNMT1 proper nuclear localization and maintenance DNA methylation in mouse embryonic stem cells. (A) UHRF1^−/− mouse ES cell lines stably expressing Flag-Myc-tagged UHRF1 wild-type or various mutants were analyzed by western blot using antibodies that recognize UHRF1. Flag-Myc-tagged UHRF1 or mutants expressed at a level similar to endogenous UHRF1 were selected for the study. β-actin was selected as a loading control. (B) Immunofluorescence analysis of DNMT1 focal staining pattern in UHRF1 wild-type, UHRF1 knockout mouse ES cells and UHRF1^−/− ES cells transfected with UHRF1-ΔUBL or UHRF1-ΔRING truncated mutant. Exogenous expression of UHRF1 and mutants was detected by Flag antibodies. Scale bars, 10 μm. (C) Immunostaining using an antibody against 5mC in control and UHRF1^−/− mouse ES cells after genetic complementation with wild-type or UHRF1-ΔUBL or UHRF1-ΔRING. The 5mC levels relative to wild-type ESCs were shown. Error bars represent ± s.e.m. (D) The DNA methylation status of LINE1 and IAP was analyzed by bisulfite sequencing in wild-type ESCs (as control), UHRF1^−/− ESCs and UHRF1^−/- ESCs stably expressing UHRF1 wild-type, or UHRF1-ΔUBL or UHRF1-ΔRING mutants. The percentage of 5mC was calculated and shown.