Dynamic phosphorylation of MCAK on Ser715 in cell cycle.
(a) Nocodazole-arrested HeLa cells were treated with BI2536 or DMSO for another 1 hr, followed by Western blot analysis with antibodies against pSer715-MCAK, MCAK, pThr210-PLK1, PLK1 and α-tubulin, respectively. (b) Images of mitotic HeLa cells treated with BI2536 or DMSO. Cell were fixed and stained with anti-pSer715-MCAK antibody (green), anti-pThr210-PLK1 antibody (red), ACA (blue) and DAPI. Insets on the right show single focal planes of the boxed regions. Scale bars, 10 μm and 1 μm (enlarged images). (c,d) HeLa cells were synchronized to G1/S boundary by a double thymidine block or prometaphase by Nocodazole treatment. Cells were then harvested and analyzed by Western blotting with the indicated antibodies. Tubulin and MCAK served as a loading control respectively in (c) and (d). (e) Double thymidine treated HeLa cells were released into fresh medium at indicated time. The harvested cells were then analyzed by Western blotting with antibodies against pSer715-MCAK, MCAK, PLK1, CyclinB and α-tubulin (loading control). (f) Images of HeLa cells stained with anti-pSer715-MCAK antibody (green), ACA (red), DAPI (blue) and anti-α-tubulin antibody. Representative kinetechores are enlarged and shown on the right. Scale bars, 10 μm and 1 μm (enlarged images).