Fig 2: PCNA degradation after DNA damage is dependent on acetylation. (A) Time course analysis of PCNA levels in the soluble and chromatin-bound pools of PCNA in quiescent, CHX-treated (25 µM) LF-1 fibroblasts after UV-irradiation (10 J/m2). Western blot analysis of PCNA, histone H3, and actin (loading controls), are shown. (B) Densitometric quantification of the soluble form of PCNA in CHX-treated and UV-irradiated (10 J/m2) quiescent LF-1 fibroblasts. PCNA levels were normalized to actin content. Mean values ± s.d. of at least three independent experiments are shown. (C) Western blot analysis of PCNA and actin (loading control) in the soluble and chromatin-bound pools in LF-1 fibroblasts incubated with non-targeting (NT) siRNA, or siRNA targeting CBP and p300 (CBP/p300), at 4 h after UV irradiation (10 J/m2). (D) Densitometric quantification of soluble PCNA (normalized to actin) in LF-1 fibroblasts incubated with non-targeting siRNA (NT), both siRNA to CBP/p300, or in single exposure, or to PCAF, in untreated (C), or 4 h after UV irradiation (10 J/m2). Mean values ± s.d. of three independent experiments are shown. (E) Western blot analysis of soluble RFP-PCNA and actin (loading control) in HeLa cells expressing wt, 2KR or 5KR mutant RFP-PCNA, before (0), or at 4 and 8 h after UV irradiation (10 J/m2) in the presence of CHX. (F) Densitometric quantification of soluble RFP-PCNA levels (normalized to actin) at 4 h after UV irradiation (10 J/m2) in HeLa cells expressing wt, 2KR or 5KR RFP-PCNA. Mean values ± s.d. of three independent experiments are shown. (G) Immunoprecipitation of RFP-PCNA with anti-RFP antibody in whole extracts from HeLa cells expressing wt, 2KR or 5KR RFP-PCNA, at 30 min after UV irradiation (10 J/m2), in the presence of MG132. Western blot analysis of immunocomplexes was performed with anti-ubiquitin antibody. RFP-PCNA levels in the input extracts are shown below.