Figure 9.

Residues 440-525 within the Linker domain are required for Mcm10 proteolysis during M-phase. U2OS cells expressing different regions 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 proteolysis of different regions of Mcm10 evaluated after release from nocodazole by immunoblotting with an anti-HA antibody has been shown in parts (A) and (C) and the flow cytometry of propidium iodide-stained DNA of stable cell-line expressing the corresponding fragment has been shown in parts (B) and (D) respectively. ASN refers to samples from asynchronously growing cells while NS points to a non-specific band that displays equal protein loading. The observed mobility of fragments of Mcm10 (fused to GFP and HA epitope tags) was as follows: LNK domain (440-607): 55 kDa; 440-471: 44 kDa; 471-525: 46 kDa; 525-607: 49 kDa; 440-525: 50 kDa; 471-607: 53 kDa; 440-607 (471-525): 53 kDa.