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Fig. 1 | Epigenetics & Chromatin

Fig. 1

From: The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

Fig. 1

High-confidence interaction partners of ZFP57 in mouse ESCs. a Western blot analysis of ZFP57 and KAP1 in wild-type (WT), Zfp57-AviTag-transfected and Zfp57-/- ESCs. Note that AviTag-ZFP57 migrates as a 60 kDa band while the endogenous ZFP57 migrates as a 50 kDa-band. b Experimental workflow of the tagged protein-mass spectrometry approach used for identification of the ZFP57 interactors. The Zfp57 cDNA was cloned into the expression vector pEF6-Avitag-GGGx2-Avitag. Zfp57-AviTag was transfected in stably BirA-expressing mouse ESCs and biotin-labelled pulled proteins were pulled down using streptavidin. Precipitated protein complexes were digested with trypsin for LC–MS/MS analysis. Proteins in common between Zfp57-AviTag-transfected and Mock-transfected BirA-expressing E14 ESCs were excluded from further analyses. Images of petri dishes and eppendorf tubes are taken from https://doi.org/10.7875/togopic.2020.104 and https://doi.org/10.7875/togopic.2022.115, respectively. BirA is depicted in light blue, biotin in red, ZFP57 in dark blue and ZFP57-interactors in grey, green, and olive. Zfp57-AviTag-transfected and mock-transfected BirA-expressing E14 ESCs are depicted in red and green, respectively. c Chord diagram showing enriched GO clusters of ZFP57 interactors. The proteins identified by LC–MS/MS analysis ordered according to their relative enrichment are shown on the left, and the enriched GO clusters are indicated with different colours on the right. The number of peptides by which proteins were recognized by LC–MS/MS analysis is displayed in gradient red (2–20 peptides). d Western blot of proteins pulled down with streptavidin in WT and Zfp57-AviTag-transfected ESCs and revealed with anti-MSH2 antibody. e Western blot of proteins pulled down with streptavidin in Msh2-AviTag-transfected ESCs and revealed with anti-KAP1 antibody. f Western blot of proteins immunoprecipitated with anti-KAP1 antibody in WT and Zfp57-/- ESCs and revealed with anti-MSH2 antibody. Input corresponds to 1% of the cell lysate used for immunoprecipitation

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Epigenetics & Chromatin

ISSN: 1756-8935