Figure 6: Cep57 interacts with Mad1.

(a) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. (b) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. (c) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. (d) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. (e) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. (f) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. (g) Schematic of truncated mutants of Mad1. (h,i) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) (h) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) (i) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. (j) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.