Fig. 4.

The RSF1-HDAC1 interaction is required for faithful chromosome segregation. a Recombinant GST-RSF1 proteins were incubated with Flag-HDAC1 expressing mitotic lysates and subjected to immunoblotting. N1: amino acids 1–627, N2: 1–871, C2: 982–1441, PHD (plant homeodomain): 628–973. b GST-RSF1 was incubated with Flag-HDAC1 deletion mutants expressing mitotic cell lysates for 1 h at 4 °C. Precipitates were subjected to immunoblotting with anti-Flag. D1: amino acids 50–482, D2: 250–482, D3: 1–325, D4: 1-430. c The RSF1-C1 WT or 5A mutant was transfected into RSF1 KO cells, and mitotic lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting. d, e RSF1 WT or KO HeLa cells were introduced with RSF1-C1 WT or 5A mutant and treated with nocodazole for 4 h. Floating mitotic cells were subjected to chromosome spread immunostaining with anti-HDAC1 or anti-H2A-pT120 and anti-RSF1 antibodies. The percentages of cells exhibiting the arm or centromeric localizations of HDAC1 proteins or H2A-pT120 level were plotted on a graph. f HeLa cells transfected with RSF1 WT or indicated mutants in RSF1 KO cells were subjected to metaphase chromosome spread and stained with Giemsa. Quantification of the percentage of premature sister chromatid separation in HeLa cells were shown. Scale bar, 5 μm