Extended Data Fig. 6: Characterization of KCTD10 self-association.
From: KCTD10 is a sensor for co-directional transcription–replication conflicts
a, representative PLA control for KCTD10 antibody used throughout this manuscript. b, co-immunoprecipitation assay in HEK293T cells expressing FLAG-KCTD10. Prior to immunoprecipitation with anti-FLAG beads, whole cell lysates were digested with benzonase or mock digested. c-d, PLA for POLR2A-KCTD10 in U2OS cells treated as with PCNAi1 (2 μM) and/or ICRF193 (100 nM). Data are presented as mean values ± SD (n = 200 cells examined over 3 independent experiments). e, co-immunoprecipitation assay in HEK293T cells with the indicated FL-KCTD10 constructs and HALO-KCTD10 treated with ICRF193 (1 μM) for 4 h. f, co-immunoprecipitation assay in HEK293T cells with the indicated FL-KCTD10 constructs and HALO-KCTD10 treated with ICRF193 (1 μM) for 4 h. g-h, PLA for HALO-FLAG in U2OS cells expressing HALO-tagged and FLAG-tagged KCTD10 with the indicated constructs as depicted in g and Fig. 3f. g, quantification of foci. Data are presented as mean values ± SD (n = 200 cells examined over 3 independent experiments). h, representative images. i-k, predicted structure of a KCTD10 dimer from Alphafold2-multimer. i, full view of the dimer. j, zoomed-in and rotated view of the dimer interface with residues of interest noted. k, alternative view of the dimer interface. l, in vitro binding assay between GST-tagged wild-type (WT) and R167A-mutant KCTD10 and FLAG-tagged KCTD10. Left, experimental schema. Right, immunoblot after GST pulldown and Coomassie stain of purified GST-KCTD10. m, co-immunoprecipitation assay in HEK293T cells transiently expressing WT and R167A-mutant FLAG-KCTD10 and treated ICRF193 (1 μM) or DMSO (4 h). Error bars indicate the mean ± SD. Scalebars = 10 μm. p-values reported from a one-way ANOVA with Bonferroni’s multiple comparisons test.
