Extended Data Fig. 8: Additional data supporting role of GSK3A in regulating cohesin unloading through WAPL, Related to Fig. 5.
From: High-throughput Oligopaint screen identifies druggable 3D genome regulators
a) Additional biological replicates of western blot of nucleoplasmic and chromatin-bound WAPL in control and GSK3A KD. Proteins labelled with HRP-linked antibodies. b) Additional biological replicates of co-immunoprecipitation of chromatin fractions in control and GSK3A KD with blotting of WAPL in SMC1A-IP. c) Quantification of WAPL normalized intensity in SMC1A-IP. *** P-value = 0.0007, two-tailed t-test. d) Additional biological replicate of GSK3A Turbo-ID. Western blots for cohesin components input lysate or lysate from with 24 h biotin incubation with either control construct (V5-BirA) or GSK3A TurboID (GSK3A-V5-BirA). Proteins labelled with HRP-linked antibodies. e) Western blot of RAD21 in chromatin fractions of control and 72 h NIPBL KD. Proteins are labelled with fluorescent antibodies. All bands from same blot, cropped for clarity. Two biological replicates represented. f) Quantification of western blot in Extended Data Fig. 6e. Protein intensity was adjusted for background, normalized to H3 volume and then normalized to the control lane for two biological replicates shown. g) Representative 3D DNA FISH images of chr22 domains after 72 h NIPBL KD, 24 h 20 µM BRD0705 treatment, or both. Dotted white line indicates nuclear edge. Scale bar nucleus, 5 µm; Scale bar spots, 1 µm. h) Fold change in mean spatial overlap between chr22:D1-D2. * P-value < 0.05, **** P-value < 0.0001, two-tailed Mann-Whitney U-test. [Alleles for Control+DMSO (n = 732); Control+BRD0705 (n = 440); NIPBL+DMSO (n = 543); NIPBL+BRD0705 (n = 373)].
