Extended Data Fig. 9: Western blot detection of centromeric proteins across 5-40% sucrose gradients and clonogenic survival assay.
From: CENP-N promotes the compaction of centromeric chromatin
a) Comparison of CENP-A (gray) and H3K9me3 (lilac) distribution in the presence of endogenous WT-CENP-N. b) CENP-A distribution in the presence of endogenous (gray) and transgenic (blue) WT-CENP-N. c) Comparison of CENP-A (blue) and CENP-N (orange) in the presence of WT CENP-N. d) Comparison of CENP-A (blue) and CENP-N (yellow) in the presence of K102A CENP-N. e). Comparison of CENP-A (pink) and CENP-N (green) distribution in the presence of 7ala CENP-N. f) Comparison of CENP-A (gray) and CENP-C (red) distribution in the presence of endogenous WT-CENP-N. g) CENP-N (orange) and CENP-C (red) distribution in the presence of endogenous WT-CENP-N h) CENP-N (yellow) and CENP-C (green) distribution in the presence of K102A CENP-N. i) CENP-N (yellow) and CENP-C (green) distribution in the presence of 7ala CENP-N. j) Representative crystal violet-stained colonies from clonogenic survival assay showing viable CENP-N AID cells with the indicated transgenic CENP-N variant treated with or without 0.1 mM IAA and doxycycline for 14 days. After treatment of 1000 seeded cells/well, surviving colonies were fixed and stained with crystal violet stain. k) Quantification of average percentage survival (average crystal violet stain intensity) of CENP-N AID cells (except CENP-N 7-ala) from 3 biological replicates normalized to untreated cells. (CENP-N 7-ala survival data is from single replicate). Data are presented as mean values± SD. l) Western blot of CENP-A and H4 histone distribution across 5–40 % sucrose gradient. Presence of CENP-A signal indicates fractions containing the centromeric chromatin whereas H4 signal represents the overall amount of chromatin loaded onto a SDS gel.
