Figure 4.

Depletion of Ssu72 causes the premature dissociation of cohesion. (A, B) HeLa cells were transfected with shRNAs against Ssu72 (shSsu72) or luciferase (shLuc; control). At 12 h post-transfection, cells were synchronized at the G1-S boundary by double-thymidine block, at the G2 phase by doxorubicin treatment, or at mitosis by nocodazole treatment. (A) Synchronized cells were separated into chromatin and non-chromatin supernatant fractions, resolved by SDS–PAGE (10%), and immunoblotted with anti-SA2, anti-Rad21, anti-phospho SA2 serine 1224 (SA2-S1224), anti-tubulin (as a non-chromatin-fraction marker), anti-histone H3 (as a chromatin-fraction marker) and anti-Ssu72 antibodies. (B) Synchronized cells were separated into chromatin and non-chromatin supernatant fractions, and cellular extracts were incubated with or without λ phosphatase (λ PP), resolved by SDS–PAGE (6%), and immunoblotted with anti-SA2, anti-Erk2 (as a non-chromatin-fraction marker) and anti-histone H3 antibodies. (C) HeLa cells were transfected as indicated, synchronized by a double-thymidine block followed by a 6-h release, and then treated with nocodazole (as indicated) to enrich for cells in the early phase of mitosis, prior to metaphase. The cells were then separated into soluble non-chromatin supernatant (Supt) and insoluble chromatin (Chro) fractions, resolved by SDS–PAGE (10%), and immunoblotted with anti-SA2, anti-Rad21, anti-Ssu72, anti-Erk2 and anti-histone H3 antibodies. (D, E) HeLa cells were transfected with siLuc, shSsu72 and siScc2 (a positive control), respectively, and synchronized at G2 and mitosis. Cellular extracts were immunoblotted with anti-Ssu72, anti-Scc2 and anti-actin antibodies. ChIP was performed with Rad21 or control IgG antibodies on cells synchronized at G2 or mitosis, and qPCR analyses were performed using primer pairs specific for the cohesin and CTCF-binding sites. The relative transcript levels were normalized with respect to that of the control siLuc in G2 phase (mean of n=3; error bars, ±s.d.).