Figure 1
The abundance of CAP-H2 is regulated by Plk1 during mitosis. (A) HeLa cells were left untreated (Asy.) or treated with nocodazole (Noc.). Mitotic arrested cells were then released for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. A ratio of CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software (Vilber Lourmat). The ratio of CAP-H2/Tubulin in asynchronous cells was defined as 1.0. Total RNA was analyzed by real-time RT-PCR with CAP-H2-specific primer. The value is normalized to GAPDH. Data represent mean ± SD from three independent experiments. (B) HeLa cells were arrested at mitosis by nocodazole with or without BI2536. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. A ratio of CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of CAP-H2/Tubulin in asynchronous cells was defined as 1.0. Total RNA was analyzed by real-time RT-PCR with CAP-H2-specific primer. The value is normalized to GAPDH. Data represent mean ± SD from three independent experiments. (C) GFP-CAP-H2 stable cell lines were arrested by nocodazole with or without BI2536. Cell lysates were analyzed by immunoblotting with the indicated antibodies. A ratio of GFP-CAP-H2 or CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of GFP-CAP-H2 or CAP-H2/Tubulin in asynchronous cells was defined as 1.0. (D) GFP-CAP-H2 stable cell lines were arrested by nocodazole with or without BI2536, and then fixed. Fixed cells were stained with anti-phospho-histone H3-Ser10. DNA was stained by DAPI. Scale bar, 20 μm. (E) The mean fluorescence of mitotic GFP-CAP-H2 was normalized by fluorescence of interphase GFP-CAP-H2. n > 30 mitotic cells per indicated condition were analyzed. Data represent mean ± SD from three independent experiments. Statistical analysis was performed with the Student’s t test (*P < 0.05).
