A“Far‐Western” blot analyses of BER protein interactions

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SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation A“Far‐Western” blot analyses of BER protein interactions. Recombinant purified human TDG (10 μg), POLβ (1 μg), APE1 (1 μg), and LIGIII (1 μg) probed with XRCC1 and XRCC1‐SUMO (20 μg) and immune‐stained for XRCC1. BImmunoblot analysis of interactions of XRCC1 and XRCC1‐SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro‐modified and in vitro‐purified 6hisXRCC1‐SUMO was coupled to Ni‐NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions. C–H(C–F and H) Scheme of pulldown experiments and immunoblot analysis of purified 6hisTDG, 6hisXRCC1/XRCC1‐SUMO and 6hisBER proteins bound to streptavidin (St) biotin‐5caC DNA substrate. (C) 0.5 μM TDGwt was incubated with biotinylated 5caC substrate (G•5caC DNA, 1 μM) and homoduplex competitor DNA (G•C DNA, 10 μM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins. (D) Schematic of biotin‐5caC DNA substrate pulldown experiment. 0.5 μM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 μM) and homoduplex competitor DNA (G•C DNA, 10 μM) as indicated and equimolar amounts of XRCC1‐SUMO (> 50% SUMOylated), POLβ and LIGIII. (E) 1 μM biotinylated 5caC substrate (G•5caC DNA) and 10 μM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1‐SUMO (50% SUMOylated) and BER proteins as indicated. (F) 0.5 μM human TDGwt protein were incubated with 1 μM biotinylated 5caC substrate (G•5caC DNA), 0.5 μM SUMO and 10 μM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO. (G) “Far‐Western” blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1‐SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1‐SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti‐TDG and anti XRCC1 antibodies as indicated. (H) Schematic of biotin‐5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG‐SUMO‐binding mutant (TDGE310Q), 6hisXRCC1/XRCC1‐SUMO and 6hisBER proteins bound to streptavidin (St) biotin‐5caC DNA substrate. 0.5 μM TDGwt or TDG‐SUMO‐binding mutant (TDGE310Q) was incubated with biotinylated 5caC substrate (G•5caC DNA, 1 μM) and homoduplex competitor DNA (G•C DNA, 10 μM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome. Source data are available online for this figure. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 38, Issue: 1, First published: 06 December 2018, DOI: (10.15252/embj.201899242)