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. 2013 May 17;288(27):19715–19725. doi: 10.1074/jbc.M112.449652

FIGURE 6.

FIGURE 6.

An excess of phosphorylated MCM7 blocked S phase entry in human normal cells. A, MCM7 interacts with Cdk2 in HL-7702 cells. The lysates from the human normal liver cell line HL-7702 were immunoprecipitated (IP) with Cdk2 antibody or normal mouse IgG and then immunoblotted (IB) with MCM7 antibody. NMS, normal mouse serum. B, MCM7 Ser-121 is phosphorylated by cyclin E/Cdk2. pCMV myc-Cdk2 AF, pCMV and myc-Cdk2 DN together with pCMV myc-cyclin E were transfected into HL-7702 cells. The cell lysates were immunoprecipitated with MCM7 antibody and then immunoblotted with MCM7-p-Ser-121 antibody. C, pCMV FLAG-MCM7-WT or pCMV FLAG-MCM7-S121A was transfected into HL-7702 cells. The total cell lysates were harvested for immunoblotting with Chk1-p-Ser-345 and Cdk2-p-Thr-14 antibodies. D, HL-7702 cells were transfected with pCMV FLAG-MCM7-WT or pCMV FLAG-MCM7-S121A, respectively. Cell lysates were immunoprecipitated with FLAG antibody and immunoblotted with MCM7-p-Ser-121 antibody. E, HL-7702 cells were transfected with pCMV FLAG-MCM7-WT or pCMV FLAG-MCM7-S121A together with pCMV GFP-H2B as the sorting marker. The cells were synchronized at G1 phase, released for 3 h, and subjected to flow cytometry analysis. **, p < 0.01.