FIG. 2.

PCNA associates with GFP-Polκ in BPDE-treated cells. (A) H1299 cells growing in chamber slides were infected with AdGFP-Polκ for 24 h. The resulting cultures were treated with BPDE, and 6 h later cells were fixed with formaldehyde. The resulting slides were probed with anti-PCNA. Bound PCNA antibody was detected with Cy3-coupled secondary antisera. PCNA and GFP-Polκ fluorescence were visualized by DeltaVision deconvolution microscopy. DeltaVision 3D object-builder software was used to generate a three-dimensional model of the two-dimensional polygons in each z section of a representative quadrant within the nucleus. The schematic depicts the degree to which green (GFP-Polκ) and red (PCNA) signals colocalize in three dimensions. (B) H1299 cells expressing GFP-Polκ (left side) or YFP-Polη (right side) were treated with 600 nM BPDE. After 6 h, chromatin fractions from the cells were analyzed directly from Polκ or Polη levels (upper blots). Alternatively, chromatin fractions were immunoprecipitated with anti-PCNA antibodies and the resulting immunoprecipitates were analyzed for PCNA and associated GFP-Polκ or YFP-Polη with anti-GFP antibodies. IB, immunoblotting.