Cell cycle–dependent regulation of exogenous FANCC but not exogenous luciferase expression.

(A) 293 FANCC E2 cells were treated with 1.3 mmol/L HU for 24 hours to synchronize cells at the G1/S boundary. Cells were then washed twice and fed with complete growth medium. Whole-cell lysates were prepared at the indicated times after release. A whole-cell extract was also prepared from a culture of asynchronous FANCC E2 cells (Control). The whole-cell extracts were subjected to Western blotting and probed using a murine anti-FANCC antibody (3A11). Identical results were obtained using a second monoclonal antibody (8F3; data not shown). Cell cycle kinetics in these experiments was identical to that shown in Figure 2B. (B) Whole-cell extracts of 293 LUC cells were prepared under similar experimental conditions. Extracts were analyzed for luciferase expression by Western blotting and probing with a rabbit anti-luciferase antiserum. The top band is a luciferase-specific band (LUC) detected only in 293 LUC cells and not in 293 WT, 293 NEO, 293 FANCC, or 293 L554P cells (data not shown). The lower 2 bands are nonspecific and are seen in 293 WT and derivative cells.