Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2014 Jun 21;42(13):8285–8296. doi: 10.1093/nar/gku522

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2014. 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/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

Figure 4. — PADI4 citrullinates DNMT3A in vitro and in vivo, via a PWWP-adjacent region. (A) PADI4 fused to GST was used in in vitro deiminase activity assays to assess citrullination of His-DNMT3A full-length (aa 1–908) fusion protein used as substrate. The presence of citrullinated DNMT3A was revealed by western blotting with anti-modified citrulline antibody. (B) PADI4 citrullinates DNMT3A within an upstream PWWP region. In vitro deiminase assays were performed as in (A), this time using several rather large (200–300 aa) fragments. This revealed the N-terminal part of DNMT3A (amino acids 1–300) as the region citrullinated by PADI4 (upper panel). In the lower panel, smaller fragments within this 1–300 region were used. Schematic representation of the various His-fusions of DNMT3A used, shown with zooming-in on the identified citrullinated sequence 197–290, which contains 5 arginines. The vertical line indicates juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. (C) Upper part, outline of the experimental procedure used to monitor citrullination of DNMT3A. U2OS cells were transiently transfected with the expression vector for wt or mutant HA-tagged PADI4. Nuclear extracts were prepared and tested for in vitro deiminase activity, using recombinant His-DNMT3A as a substrate. Coomassie staining was used as loading control. (D) Citrullination of endogenous human DNMT3A by PADI4 in vivo. As described in Figure 1C, lysates of untransfected U2OS cells were immunoprecipitated with anti-DNMT3A or control (IgG) antibodies. Endogenous and modified DNMT3A (citrullinated DNMT3A) were this time revealed, respectively, by western blotting with anti-DNMT3A and anti-modified citrulline antibodies. % of Input (20%) is shown. (E) Decreased citrullination and reduced expression of endogenous DNMT3A in PADI4-knockout MEFs. Nuclear lysates of wt and PADI4-knockout MEFs were immunoprecipitated with anti-DNMT3A. Endogenous and modified DNMT3A (citrullinated DNMT3A) were revealed by western blotting with anti-DNMT3A and anti-citrulline antibodies, respectively. PADI4 expression in wt versus knockout MEFs is shown after western blotting with anti-PADI4.