Mapping of the agnoprotein and PCNA regions required for interaction. (A) Amino acid sequences of the agnoproteins of BKPyV, JCPyV, and SV40 (top part) and schematic representation of BKPyV wild-type agnoprotein (agno WT) and truncated peptides (bottom part). The conserved “PIP box like sequence” is indicated. The regions encompassing peptides 1–37, 15–45, and 8–66 are indicated by arrows. The number at the end of the amino acid sequence refers to the number of residues in the protein. The name of the peptides corresponds to the residue numbers. PIP signifies the PCNA Interaction Protein box and the mutation QRIFIF into ARAFIA is symbolized by an X. (B) GST-pull down of purified GST-PCNA fusion protein and purified wild-type agnoprotein or agnopeptides. GST-PCNA (lanes 1, 3, 5, 7, 9) was mixed with full-length agnoprotein (lane 3) or agnoprotein peptide fragments comprising residues 1–37 (lane 5), residues 15–45 (lane 7) or residues 15–45 with mutated PIP motif (lane 9), respectively. The presence of full-length agnoprotein or peptides and PCNA was monitored by immunoblotting using antibodies against agnoprotein and PCNA simultaneously. The band representing agnoprotein dimer (lanes 2 and 3) is indicated. The additional weaker bands in lanes 3, 5, 7 and 9 probably represent degradation products. (C) GST-pull down experiments using purified GST-agnoprotein. Left panel: GST-agnoprotein was incubated with different mutants of PCNA (lanes 3, 5, 7) or GST-PCNA (lane 9) and complexes were pulled down. The presence of agnoprotein and PCNA was investigated by immunoblotting using antibodies against agnoprotein and PCNA, respectively. M = molecular mass marker in kDa. Right panel: Coomassie blue staining of purified GST (lanes 2 and 6), GST-agno (lanes 3 and 7) and GST-PCNA (lanes 4 and 8). Lanes 1 and 5: Precision Plus Protein Dual Color Standards (BioRad) marker.