Figure 6

Cell cycle degradation of Mcm10 is independent of APC. (A) HeLa cells were transfected on three consecutive days with APC3 or control GL2 siRNA. After the third transfection, the cells were arrested with nocodazole for 15 h and were harvested after release from nocodazole (Noc) for analysis of Mcm10 protein, along with asynchronous cells (ASN). Samples were also taken 2 h after release from nocodazole (2 h) (B) The decrease of APC3 protein after RNAi was confirmed by immunoblotting with anti-APC3 antibody. NS points to a non-specific band that displays equal protein loading in different lanes. (C) The decrease of APC3 mRNA was confirmed by RT-PCR and the numbers indicate the relative intensity of the mRNA. (D) Flow cytometry of propidium iodide-stained DNA of HeLa cells, as described in (A) confirms a M-phase block and release after siRNA depletion. The observed mobility of endogenous APC3 was 73 kDa.