Figure 4. Direct interaction between PCNA and DDB2. (A) The analysis of interaction between proteins has been performed by immunoprecipitation, as reported in “Material and Methods”. DDB2 recombinant protein was present in each sample: p21^wt-His protein was added in sample 1, PCNA recombinant protein was added in sample 2, both p21 and PCNA recombinant proteins were added in sample 3, and both PCNA and p21 mutant proteins were added in sample 4. (B) Identification and phylogenetic comparison of PIP-box in the DDB2 amino acidic sequence. The sequence is conserved from zebrafish to man. (C) Sequence alignment of the PIP-box motif of DDB2 with conserved PIP-box motifs of p21, another PCNA-interacting protein. The consensus PIP-box sequence is indicated. Underlined amino acids have been mutated in DDB2 gene. (D) In vitro binding assay of DDB2^wt-PCNA (lane 1 and 3) and DDB2^mut-PCNA (lane 2 and 4). On the left panel, immunoprecipitation (Ip) was performed with anti-DDB2 antibody; on the right, with anti-PCNA antibody. Immunoprecipitation was performed as described in “Materials and Methods”.