Fig. 6. p21 does not displace pol from binding to PCNA after UV-C DNA damage. (A) Immunoprecipitation (Ip) was performed on detergent-soluble (S), or chromatin-bound fraction (Cb) obtained from LF1 fibroblasts irradiated or not with UV-C (10 J/m²), and harvested after 30 minutes. Samples were immunoprecipitated with anti-p21, or with anti-p125 (pol ) polyclonal antibodies, or with purified rabbit immunoglobulins (Ig) for specificity control. The immunoprecipitated material was analysed by western blot for the presence of PCNA, pol (p125 subunit), and p21. The position of each protein is shown together with Ig heavy chains (Ig h). Fractionated extracts (Input) were loaded (1/30 and 1/15 for S and Cb fractions, respectively) together with recombinant PCNA (PCNAr), and analysed by western blot for pol , PCNA, p21, and actin as a loading control. (B) Immunoprecipitation (IP) was performed on HeLa chromatin-bound extracts with anti-GFP antibody. Cell extracts were obtained from cells expressing pEGFP (GFP), or p21-GFP, irradiated or not with UV-C (10 J/m²) and harvested at times indicated below each panel. Western blot analysis of PCNA and pol , was performed on immunoprecipitated material. The position of each protein is shown together with Ig heavy chains (Ig h). Chromatin-bound extracts (Input) were loaded (1/15) on a parallel gel for western blot analysis of pol , PCNA, p21-GFP, and actin as a loading control.