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. 2020 Mar 6;11:1222. doi: 10.1038/s41467-020-15006-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Immunoprecipitation of endogenous DNMT1 from whole-cell lysates of wild-type J1 (WT), Dnmt1 KO (D1KO), Uhrf1 KO (U1KO), Paf15 K15R (K15R), Paf15 K24R (K24R), and Paf15 K15/24R (KRKR) mESCs using an anti-mDNMT1 nanobody. Bound fractions were subjected to immunoblotting with anti-mDNMT1 and anti-mPAF15 antibodies. The anti-mDNMT1 blot and Ponceau staining are shown as loading controls. b Immunoprecipitation of endogenous mPAF15 from WT and KRKR mESC nuclear extracts using an anti-mPAF15 antibody. Bound fractions were subjected to immunoblotting with anti-mDNMT1 and anti-mPAF15 antibodies. The anti-mDNMT1 blot and Ponceau staining are shown as loading controls. c Schematic of the fluorescent-3-hybrid (F3H) assay for the in vivo determination of protein–protein interactions. GFP-tagged bait protein is immobilized at an array of Lac operator (LacO) sequences by a GFP-binding protein (GBP) coupled to the lac repressor (LacI). When the GFP-tagged bait protein does not interact with the prey protein, only a GFP signal is visible at the LacO locus, whereas a yellow spot (combination of GFP and mCherry signal) is visible at the LacO locus in the case of a positive interaction. d, e F3H assay for a BHK cell-based analysis of ubiquitylation-mediated recruitment of mPAF15 to mDNMT1. d Cells containing a stably integrated lacO array were transfected with the GBP-LacI, a GFP-tagged bait (GFP-mDNMT1 or GFP), and an mCherry-tagged prey (mCherry-mPAF15 wild-type (WT) or mCherry-mPAF15 K15R/K24R double mutant (KRKR)). Line intensity profiles for GFP and mCherry in the respective spots are shown below the confocal images. Scale bar, 10 μm. e Quantification of the F3H assay. Background subtracted mCherry/GFP ratios within the spots were normalized to the control and plotted with n = 45 from 3 independent replicates (per replicate, n = 15). In the boxplots, horizontal black lines within boxes represent median values, boxes indicate the upper and lower quartiles, and whiskers indicate the 1.5× interquartile range. Statistical significance was determined using Student’s t test. Source data are provided as a Source Data file.