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Figure 7


Fig. 7. p21 recruitment to DNA repair sites requires interaction with PCNA. HeLa cells were transfected with HA-tagged constructs for expression of wild-type p21 (p21HAwt) or a mutant form (p21HAmt) unable to bind PCNA (p21PCNA–). (A) 24 hours after transfection, cells were exposed to local UV irradiation (15 J/m2) through filters with 3 µm pores, extracted in situ 30 minutes later and fixed for immunofluorescence staining with anti-HA (green fluorescence) or anti-PCNA (red fluorescence) antibody. Confocal sections of each signal, together with the merged images, are shown. Bars, 5 µm. (B) Immunoprecipitation was performed with anti-HA antibody on detergent-soluble (soluble), and chromatin-bound (chrom.) fractions obtained from non-transfected (ntr) cells, and from cells expressing p21wt (wt), or p21PCNA– (mt) proteins. Immunoprecipitated material was analysed by western blot with anti-pol {delta}, anti-PCNA, and anti-HA antibodies. The position of each protein is shown together with Ig heavy (Ig h), and light (Ig l) chains. For detergent-soluble and chromatin-bound extracts, 1/30 and 1/15 respectively, were loaded (Input) on a parallel gel for western blot analysis of pol {delta}, PCNA, p21HA-tagged proteins, and actin as a loading control.





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