Fig. 3: CoREST1^NT2-HDAC2 is the common binding entity for KBTBD4^R313PRR and KBTBD4^WT-UM171.

a For crosslinking experiments, 2.5 μM of indicated proteins were mixed and incubated with 4 mM BS³ crosslinker. The reactions were terminated by laemmli buffer and then analyzed in parallel with uncrosslinked controls on an SDS-PAGE gel, followed by Coomassie staining. Crosslinked or uncrosslinked species were labelled alongside the gel image. b Unlabelled CoREST1^NT2-HDAC2 was titrated at indicated concentrations into 40 nM fluorescently-labelled KBTBD4 variants, with or without 25 μM UM171, for MST measurements. The apparent K_D values of CoREST1^NT2-HDAC2 binding to KBTBD4^R313PRR and KBTBD4^WT-UM171, respectively, were determined to be 0.75 µM and 15 µM, while negligible binding was observed to KBTBD4^WT alone. c Unlabelled UM171 was titrated at indicated concentrations into 40 nM fluorescently-labelled KBTBD4^WT with 5 μM CoREST1^NT2-HDAC2 for MST measurements (apparent K_D = 14 µM). All MST experiments were performed in triplicates. Data points and error bars represent the means and standard deviations. Source data are provided in the Source Data file. d Various E2 enzymes were mixed with CoREST1^NT2-HDAC2, KBTBD4^R313PRR, neddylated CUL3, E1 and ubiquitin, and incubated in the presence of ATP and MgCl₂ to measure ubiquitylation. The reactions were analyzed on an SDS-PAGE gel and visualised by Coomassie stain. Ubiquitylated HDAC2 and KBTBD4^R313PRR showed higher molecular weight ladders as labelled in the figure. e Immunoblots of in vitro ubiquitylation reaction mixtures using UBE2D1 E2 enzyme. KBTBD4 variants and UM171 were added to the reaction mixtures as indicated. The ubiquitylation experiments were performed at least twice with similar results. Source data are provided in the Source Data file.