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. 2016 Jul 20;2(7):e00131. doi: 10.1016/j.heliyon.2016.e00131

Fig. 8.

Fig. 8

Impact of overexpression of INCENP-S894A/D mutants on the mitotic checkpoint. (A) Phosphorylation of S894 on INCENP is dispensable for the interaction with Aurora-B. HEK293T cells were co-transfected with 3FLAG-tagged Aurora-B-WT (wild-type) or -KD (kinase dead) and 6Myc-tagged INCENP-WT, -S894A, or -S894D. Cell lysates were immunoprecipitated with anti-Myc antibody (IP), followed by western blotting with anti-Myc and anti-FLAG antibodies. (B) HeLa-S3 cells were transiently transfected with 6Myc-tagged INCENP-WT, -S894A, -S894D, or vector alone, and then treated with nocodazole or taxol. Cell lysates were immunoblotted for phosphorylated S10-Histone H3 (pS10-H3), Histone H3, pS7-CENP-A, CENP-A, pan phospho-Aurora-A/B/C (for detection of Aurora-B–pT232), Aurora-B, cyclin B1, Myc-tag, and α-tubulin antibodies. The level of pS10-H3 was normalized against the level of total Histone H3. (C) Flow cytometry analysis. HeLa-S3 cells were transiently transfected with 6Myc-tagged INCENP-WT, -S894A, -S894D, or vector alone, and then treated with or without nocodazole or taxol as in B. Synchronized and asynchronous cells were stained with propidium iodide, followed by flow cytometry to generate cell cycle profiles. See Fig. S4 for uncropped western blot images in Figs. 8A, B. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.