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. 2015 Dec 9;6:10076. doi: 10.1038/ncomms10076

Figure 3. PLK1 phosphorylation of PCNT in vivo.

Figure 3

(a) HeLa cells were synchronously released from G1/S phase with the double thymidine block method and collected every 2 h for the immunoblot and immunostaining analyses. (b) The cells were subjected to immunoblot analyses with the PCNT, cyclin B1 and GAPDH antibodies. (c) Co-immunostaining analysis was performed with the centrin-2 antibody (CETN2, red) along with the PCNT and phospho-PCNT (pS2259PCNT and pS1241PCNT) antibodies (green). Scale bar, 10 μm. (d) Centrosomal intensities of PCNT and phospho-PCNT were densitometrically determined and shown with the box-and-whisker plot. n=153 per group in three independent experiments. (e) Co-immunostaining analysis of mitotic HeLa cells was performed with indicated antibodies. The mitotic stages were determined with 4,6-diamidino-2-phenylindole-staining patterns. Scale bar, 10 μm. (f) Centrosomal intensities of PCNT and phospho-PCNT were determined in cells at specific mitotic stages. n=153 per group in three independent experiments. (g) M-phase-arrested HeLa cells were forced to exit mitosis with ZM447439 (ZM). At indicated time points, the cells were subjected to immunoprecipitation (IP) followed by immunoblot analyses with antibodies specific to PCNT, pS2259PCNT, PLK1, cyclin B1 and GAPDH. (h) The M-phase-arrested HeLa cells were treated with BI2536 for 3 h and subjected to IP followed by immunoblot analyses with indicated antibodies.