Figure 3. The TFIIH complex is required for the enrichment of condensins on chromosomes.

(A) Representative immunofluorescence images of Xenopus sperm nuclei incubated with DMSO or triptolide (TPL) treated extracts for 180 min. Chromatids were labeled with Hoechst and anti-XPB, anti-CAP-D3 (condensin II), anti-CAP-C (condensin I & II), anti-CAP-E (condensin I and II), anti-SUPT16H (FACT complex), or anti-Topo II antibodies. Images of individual chromatids are shown in insets. (B) Quantification of fluorescence intensity of indicated proteins from experiment in (A), normalized to DMSO. n = 50 structures for each condition. Error bars represent SD, and asterisks indicate a statistically significant difference (*, p < 0.001), NS indicates no statistical significance. A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment.

Figure 3—figure supplement 1. The TFIIH complex is required for the enrichment of condensins on chromosomes.

(A) Left: Representative immunofluorescence images of Xenopus sperm nuclei incubated with Mock or CAP-E depleted extracts for 180 min. Chromatids were labeled with Hoechst and anti-XPB antibodies. Right: Quantification of fluorescence intensity of XPB on chromatin, normalized to Mock depleted condition. n = 24 structures for each condition. Error bars represent SD, and ‘NS’ indicates no statistically significant difference (p > 0.001). A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment. (B) Western blot for indicated proteins that copurify with anti-XPB or IgG beads isolated from HSS extracts. IP, immunoprecipitate. (C) Western blot for indicated proteins that copurify with anti-CAP-E or Mock-treated beads isolated from HSS extracts. For condensin subunit detection, the blot was probed simultaneously with the indicated antibodies. Beads were coupled to either anti-CAP-E or pre-immune serum (“Mock”) and processed as indicated in the methods. IP, immunoprecipitate. (D) Representative immunofluorescence images of Xenopus sperm nuclei incubated with HSS extracts for 180 min. Chromatids were labeled with Hoechst and co-stained with anti-XPB and anti-CAP-E antibodies. Images of three representative individual chromatids are shown.