Figure 2.

Cell cycle-regulated degradation of Mcm10 is not dependent on APC recognition motifs: KEN and destruction box. (A) Anti-Mcm10 antibody recognizes the mitotic forms of Mcm10. U2OS cells expressing the NTD+ID domain of Mcm10 (fused to GFP and HA epitope tags) were arrested in M-phase using nocodazole (0 h) and then harvested at the indicated time-points after release into drug-free medium. The levels of the NTD+ID domain of Mcm10 were independently evaluated by anti-HA (bottom panel) and anti-Mcm10 (middle panel) antibody. The same samples were probed with anti-Mcm10 antibody (Ab [N]) to display the levels of endogenous Mcm10 (top panel). ASN refers to samples from asynchronously growing cells. The antibody used for immunoblotting and the protein detected by it have been listed on the left and right sides of a particular western blot. The observed mobility of endogenous Mcm10 and NTD+ID domain (fused to GFP and HA epitope tags) was 110 kDa and 97 kDa respectively. (B) U2OS cells expressing HA-tagged full length Mcm10 were arrested in M-phase using nocodazole (0 h) and then harvested at the indicated time-points after release into drug-free medium. The levels of endogenous Mcm10 and HA-tagged full length Mcm10 were evaluated at different time points by anti-Mcm10 (top panel) and anti-HA (middle panel) antibody. NS points to a non-specific band that displays equal protein loading. The observed mobility of HA-tagged FL Mcm10 was approximately 115 kDa. (C) U2OS cells expressing wing-helix (WH) fragment of Mcm10 were arrested in M-phase using nocodazole (0 h) and then harvested at the indicated time-points after release into drug-free medium. The levels of endogenous Mcm10, WH fragment and cyclin B were evaluated at different time points after release from nocodazole. The observed mobility of WH fragment of Mcm10 (fused to GFP and HA epitope tags) was approximately 50 kDa. (D) The flow cytometry of propidium iodide-stained DNA of stable cell-line expressing WH fragment has been shown. The colored key denotes cells obtained at different time-points. (E) Immunofluorescence with HA antibody displays that the WH fragment of Mcm10 tagged with a nuclear localization signal is directed to the nucleus.

Kaur et al. BMC Cell Biology 2010 11:84   doi:10.1186/1471-2121-11-84
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