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. 2010 Oct 18;191(2):313–329. doi: 10.1083/jcb.201003090

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© 2010 Malureanu et al.

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Figure 5. — Cyclin B1 is de novo synthesized during normal mitosis. (A) Cdc20^+/+ mitotic MEFs increase cyclin B1 levels in response to MT depolymerization but not when protein synthesis is blocked with CHX. Cells were either untreated or treated with 100 ng/ml nocodazole (Noc) or 100 ng/ml nocodazole + 1 µg/ml CHX for 15 min. (B) Quantification of cyclin B1 signals (n > 10 cells per group). *, P < 0.02 versus prophase (P), prometaphase (PM), and metaphase (M); **, P < 0.001 versus nocodazole + CHX-treated cells (by unpaired t test). (C) Extracts of mitotic HeLa cells treated for 15 min with 100 ng/ml nocodazole or 100 ng/ml nocodazole and 1 µg/ml CHX analyzed for the indicated proteins by Western blotting. (D) Cdc20^+/+ metaphases in which de novo protein synthesis is blocked by CHX show decreased cyclin B1 immunofluorescence. Cells were treated with 1 µg/ml CHX 15 min before fixation. (E) Quantification of endogenous cyclin B1 signals (n = 10 Cdc20^+/+ MEFs/group). *, P = 0.0366 versus untreated cells (by unpaired t test). (F) As in D but shown for HeLa cells. (G) Quantification of endogenous cyclin B1 signals (n = 10 HeLa cells/group). *, P = 0.0005 versus untreated cells (by unpaired t test). Error bars represent SD. Bars, 10 µm.