Figure 4.

Accumulation of cyclin A during G₁ phase leads to compromised chromatin loading of MCM4 in FBXO31-depleted cells. A, NS or shFBXO31-expressing MCF7 cells were synchronized using nocodazole for 16 h. Synchronized cells were then released and collected at the indicated time points. Whole-cell protein extracts were immunoblotted to probe for the indicated proteins. B, quantitation of cyclin A expression from A. Cyclin A expression was normalized with respective loading control tubulin at the corresponding time points and then normalized to 100% at each time point for NS cells. Then, cyclin A levels in shFBXO31-expressing cells at the indicated time points were quantified with respect to corresponding NS cells. Error bars represent S.E. from three independent experiments. C, immunoblot monitoring the interaction between FBXO31–cyclin A and CDH1–cyclin A at the endogenous levels at different phases of the cell cycle. MCF7 cells were synchronized using nocodazole for 16 h. Synchronized cells were then released and allowed to grow in fresh medium until harvested. MG132 (10 μm) was added to the medium for 2 h before harvesting the cells at the indicated time points. Whole-cell protein extracts were immunoprecipitated with the indicated antibodies. The immunoprecipitates and input protein extracts were immunoblotted and probed for the indicated proteins. D, MCF7 cells expressing either NS or FBXO31 shRNA were transfected with FLAG–cyclin A in combination with either His–Lys-11–only ubiquitin or His–Lys-48–only ubiquitin for 24 h, and transfected cells were then incubated with 10 μm MG132 for 2 h. Cells were then harvested, and whole-cell lysates were incubated with Ni-NTA beads. The pulled down fractions and input protein lysates were immunoblotted for the indicated proteins. E, immunoblot (IB) analysis monitoring MCM4 level in chromatin and nonchromatin fractions during mitosis and G₁ phase of NS, FBXO31KD, and FBXO31–cyclin A knockdown cells. Error bars where shown represent S.E. from three independent experiments, and n.s. represents nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.