Figure 4.

DDK is important for mediating Rad18 S434 phosphorylation in intact human cells. (A) Partial knockdown of Cdc7 achieved using siRNA. Replicate plates of H1299 cells were transfected with siRNA against Cdc7 (siCdc7) or nontargeting control RNA duplexes (siCon), or were left untransfected. Cell lysates prepared 24 h and 48 h after transfection were analyzed for Cdc7 expression using SDS-PAGE and immunoblotting. A minor nonspecific band (NS) recognized by the Cdc7 antibody serves as a loading control. (B) Partial Cdc7 depletion does not affect numbers of S phase cells. Replicate plates of H1299 cells were transfected with siCdc7 or siCon RNA duplexes. 24 h and 48 h after transfection, cells were incubated with 10 µM BrdU for 1 h. Levels of BrdU incorporation were determined by anti-BrdU staining and FACS analysis. (C) Effect of partial Cdc7 or Dbf4 depletion of Rad18 S434 phosphorylation. H1299 cells were transfected with siRNA against Cdc7 or Dbf4 (or nontargeting control RNA duplexes). Extracts from the resulting cells were immunoprecipitated with anti-HA and analyzed by SDS-PAGE and immunoblotting with anti-Rad18 (pS434) and anti-HA. Input fractions were normalized for protein and analyzed by SDS-PAGE and immunoblotting with anti-Cdc7 or anti-Dbf4 antibodies to test for knockdown efficiency. Molecular mass is indicated in kilodaltons next to the gel blots.