Fig. 2: RSF1-dependent PLK1 deposition is required for the Aurora B kinase activity. | Nature Communications

Fig. 2: RSF1-dependent PLK1 deposition is required for the Aurora B kinase activity.

From: Spatiotemporal coordination of the RSF1-PLK1-Aurora B cascade establishes mitotic signaling platforms

Fig. 2

a The HeLa cell lysates were obtained from mitotic shake-off after treatment with nocodazole for 16 h in RSF1 siRNA-expressing cells. Western blot analysis of chromatin fraction was immunoblotted for indicated antibodies. TopoII-α was used as a marker for chromatin fraction. b Control or RSF1 shRNA mitotic cell lysates were immunoprecipitated with anti-PLK1 antibody and immunoblotted with indicated antibodies. c GFP-INCENP or HA-Aurora B plasmid was transfected into RSF1 KO cells and treated with nocodazole for 16 h. Mitotic cell lysates were subjected to immunoblot analysis with indicated antibodies. d Exogenously transfected with Myc-Plk1 WT or CA into RSF1-depleted HeLa cells and mitotic cells were obtained with nocodazole treatment for 16 h. The lysates were processed for immunoblotting analysis using indicated antibodies. e Myc-PLK1 WT, KD, or HA-Aurora B expression vectors were introduced into HeLa cells, stably expressing RSF1 shRNA, and then mitotic cells were obtained with nocodazole treatment. The mitotic cells were stained with anti-pAurora B T232 (green) and ACA (red) antibodies, and DAPI was used to stain the nuclei (blue). The graph represents mean ± SEM from three independent experiments; shCtrl n = 28, shRSF1 n = 34, shRSF1 + PLK1 WT n = 37, shRSF1 + PLK1 KD n = 29, and shRSF1 + HA-Aurora B n = 24 cells. **p < 0.005 vs. control shRNA, ***p < 0.001 vs. pcDNA in shRSF1 by two-sided unpaired Student’s t-test. Scale bar, 5 μm. Data of a, c, d are representative of three independent experiments. Data of b are representative of two independent experiments. Source data are provided as a Source Data file.

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