Fig 1.

BPLF1 interacts with PCNA both in vivo and in vitro. (A) In an in vivo interaction, immunoprecipitations (IPs) were performed on lysates from 293T cells expressing BPLF1 1-246 followed by Western blotting (WB) for endogenous levels of PCNA. Western blotting of lysates detected PCNA, BPLF1 1-246, and BPLF1 C61S. The relative amounts of constituents are shown by densitometry beneath each row. Approximately 10% of BPLF1 C61S bound to PCNA (when total expression levels of BPLF1 1-246 and BPLF1 C61S are factored in) with WT BPLF1 binding set at 100% (1.0 is equivalent to 100%). (B) In an in vivo interaction, FLAG-tagged BPLF1 1-246 and HA-tagged PCNA were overexpressed in 293T cells, and IP was performed with FLAG antibody. HA-PCNA and FLAG-BPLF1 1-246 were present in the reaction mixtures as shown in the middle and bottom panels. Mixing of lysates from samples represented by lanes 1 to 4 demonstrate that interaction occurred in the cells and not in the lysates (lane 5). Lysates from the indicated samples (lanes 5 to 8) were mixed as indicated prior to IP (e.g., lane 5 contains the samples from lanes 1 and 2 mixed together). The middle and bottom panels show the presence of BPLF1 and PCNA. The position of the IgG band is indicated by black arrows to the right of the panels. (C) In an in vitro interaction, BPLF1 1-246 and PCNA were purified from E. coli and incubated together for 30 min at 25°C. IPs were performed with BPLF1 antiserum and probed for PCNA. PCNA and BPLF1 were present in the reaction mixtures as shown in the middle and bottom panels.