Figure 1

BKPyV agnoprotein and PCNA interact in vitro and in vivo. (A) In vitro pull down experiment shows direct interaction between PCNA and agnoprotein. Purified agnoprotein, PCNA or GST-PCNA fusion protein were incubated alone or together in PBS (buffer) or with lysate of HEK293 cells (cell lysate) at 4°C for 1 h. Complexes were immunoprecipitated with PCNA antibodies (IP:anti-PCNA; lanes 1–12) or with antibodies against agnoprotein (IP:anti-agno; lanes 13–24). Samples were run on gel and western blot was performed with antibodies against PCNA (WB:anti-PCNA) or against agnoprotein (WB: anti-agno) as indicated. I = input, P = precipitate. The position of GST-agno, agnoprotein, GST-PCNA, purified PCNA and endogenous PCNA are indicated by arrows. The arrow with dashed line probably represents agnoprotein dimers (see also Figure 2B). (B) Co-immunoprecipitation of agnoprotein and PCNA. HEK293 cells were transfected with following plasmids: lanes 1 and 2: pRcCMV-agno plus pFLAG-CMV-2-PCNA, lanes 3 and 4: pRcCMV-agno plus pFLAG-CMV-2, lanes 5 and 6: pRcCMV plus pFLAG-CMV-2. Lanes 1, 3, and 5: input; lanes 2, 4, and 6: immunoprecipitates (IP). Protein complexes were precipitated with antibodies against PCNA and the presence of polδ, PCNA and agnoprotein was examined with antibodies against these proteins. (C) FRET measurements of the interaction between ECFP-PCNA (donor) and EYFP-agnoprotein (acceptor) fusion proteins in A375 cells by acceptor photo bleaching. FRET efficiency (FRET signal %) is calculated by fluorescence before (prebleach) and after bleaching (postbleach) and shown by the colour code bar. Control experiments with ECFP plus EYFP-agno and ECFP-PCNA plus EYFP were included.