FIG. 5.

Association between Rad18 and Polκ. (A) H1299 cells were infected with AdGFP-Polκ or AdYFP-Polη individually or in combination with AdHA-Rad18. After 24 h, some cultures were treated with BPDE. Chromatin fractions from the resulting cells were solubilized. Aliquots of total chromatin fractions were analyzed directly for expression of HA-Rad18, GFP-Polκ, and YFP-Polη by SDS-PAGE and immunoblotting (input). The remainder of each chromatin fraction was immunoprecipitated with anti-GFP antibodies. The anti-GFP immune complexes were resolved by SDS-PAGE and probed with anti-GFP (to verify IP of GFP and YFP TLS polymerase fusion proteins) or with anti-HA (to test for associated Rad18). (B) H1299 cells were infected with AdGFP-Polκ individually or in combination with AdHA-Rad18. At 24 h later, some cultures were given 600 nM BPDE for 6 h. The distribution of GFP-Polκ in the resulting cells was determined by deconvolution microscopy. (C) H1299 cells were infected with Ad GFP-Polκ and AdHA-Rad18. After 24 h, some cultures received 600 nM BPDE for 6 h. The resulting cells were fixed and stained with anti-HA antibodies as described in Materials and Methods.