Fig. 5. The replication fork proteins RPA and PCNA, but not nuclear lamins, colocalize with BrdU-labeled nascent DNA within replication foci. HeLa and WI38 cells were pulse-labeled with BrdU, extracted with Triton X100 to remove soluble proteins and fixed with freshly prepared formaldehyde. (A-C) One aliquot of the cells was treated with HCl and then stained simultaneously with lamin B- (green) and BrdU-specific (red) antibodies. A second aliquot was first stained for lamin B [green (D-G)] or lamin A/C [red (J-Q)], the cells were fixed again with formaldehyde, then treated with HCl and stained for BrdU (red in D-F and green in J-Q). (H) The same protocol was used to stain for PCNA (red) and BrdU (green). (I) A fourth aliquot of cells was stained directly with anti-PCNA (red) and anti-RPA (green) antibodies. Results obtained with HeLa cells are shown in panels A-I. Identical results were obtained with WI38 cells.