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. 2015 Aug 10;6:7904. doi: 10.1038/ncomms8904

Figure 1. RSF1 localizes at mitotic kinetochores and directly interacts with PLK1.

Figure 1

(a) RSF1 knockout (KO) HeLa cells were transfected with pcDNA or RSF1-V5. Floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to chromosome spread immunostaining. Immunofluorescence images of human epithelial RPE1 and HeLa cells are shown: RSF1 (green), ACA (red) and 4,6-diamidino-2-phenylindole (blue). Scale bar, 5 μm. (b) Chromatin fractions of asynchronously growing HeLa (I, interphase) and paclitaxel-arrested HeLa cells (M, mitotic phase) were obtained after centrifugation and washing with 0.5 M NaCl. Proteins were eluted as soluble fractions and chromatin-bound fractions were analysed by immunoblotting with the indicated antibodies. Topo IIα and GAPDH were used as controls for chromatin and soluble fractions, respectively. (c) In vitro binding assays: recombinant RSF1-V5 was incubated with immunopurified Myc-PLK1 or HA-Aurora B for 2 h at 4 °C. RSF1-V5 bound to immobilized Myc-PLK1 or HA-Aurora B was detected by immunoblotting. Recombinant RSF1-V5 was stained with Coomassie blue. (d) Recombinant RSF1-V5 was incubated with recombinant His-PLK1 purified from insect cells, and His-PLK1 bound to immobilized RSF1-V5 was detected by immunoblotting. Recombinant RSF1-V5 and His-PLK1 were visualized by Coomassie blue staining. (e) Recombinant GST-RSF1 proteins were incubated with Myc-PLK1 expressing mitotic lysates and subjected to immunoblotting. N1: amino acids 1–627, N2: 1–871, C2: 982–1441 and PHD (plant homeodomain): 628–973. (f) Immunopurified Flag-PLK1 was incubated with recombinant RSF1-V5. WT, wild type; KD, kinase dead; N, amino acids 1–401; C, amino acids 350–603. (g) Recombinant GST-RSF1 proteins were incubated with Flag-tagged PLK1 WT, PLK1-PBDW414F or PLK1-PBDH538A/K540M expressing mitotic lysates and subjected to immunoblotting. Recombinant GST-RSF1 was stained with Coomassie blue. (h) In vitro binding assays: recombinant RSF1-V5 was incubated with immunopurified Flag-PLK1 WT or Flag-PLK1 PBD mutants for 2 h at 4 °C. RSF1-V5 bound to immobilized Flag-PLK1 WT or Flag-PLK1 PBD mutants was detected by immunoblotting. Recombinant RSF1-V5 was stained with Coomassie blue. See full blots Supplementary Fig. 8.