Endogenous Mcm10 levels are low during mitosis. U2OS cells were blocked at the mitotic phase with nocodazole for 16 h and were either harvested immediately (0 h) or released in nocodazole-free medium and then harvested at the indicated time-points and used for immunoblotting and flow cytometry. (A) Levels of endogenous Mcm10 were evaluated by Ab (N) and Ab (FL) antibody. ASN refers to samples from asynchronously growing cells while NS points to a non-specific band that displays equal protein loading in different lanes. (B) Flow cytometry of propidium iodide-stained DNA of U2OS cells, as described in (A), show an M phase block and release. The colored key denotes cells obtained at different time-points. (C) and (D) U2OS cells were blocked in mitosis with 0.5 μg/ml colchicine, 0.10 μg/ml nocodazole, 0.3 μg/ml taxol or 0.2 μg/ml vincristine for 16 h and analyzed for levels of Mcm10 (C) and DNA content (D) by immunoblotting and flow cytometry respectively. The observed mobility of endogenous Mcm10 and cyclin B was 110 kDa and 55 kDa respectively.
Kaur et al. BMC Cell Biology 2010 11:84 doi:10.1186/1471-2121-11-84