Figure 3. Mapping of the MCV LT and Brd4 interaction sites.
A. Schematic diagrams of full-length MCV LT protein and truncation mutants used for mapping. All MCV LT fragments were expressed from a pcDNA4C plasmid encoding an Xpress epitope tag. B. Mapping the Brd4 binding site on MCV LT. 293T cells were transfected with full-length MCV LT or a truncation mutant construct. Nuclear extracts Co-IPed with normal rabbit IgG (IgG) or a Brd4 antibody (Brd4) were immunoblotted with Xpress (LT) or Brd4 antibodies. C. Schematic diagrams of full-length human Brd4 protein and truncation mutants used for mapping. All Brd4 fragments were expressed from a pcDNA4C plasmid as Xpress tagged proteins. D. Mapping the MCV LT binding site on Brd4. 293T cells were co-transfected with a Brd4 mutant construct and either the FLAG-tagged pOZN vector (V) or pOZN-MCV LT (LT). Nuclear extracts IPed with anti-FLAG beads were immunoblotted with Xpress (Brd4) or LT antibodies. Arrowheads mark the positions of the Xpress tagged Brd4 mutants. Asterisks to the right of the blots mark the positions of IP antibody heavy and light chains. For all IPs, 20 µg nuclear protein input and 20% of Co-IPed samples were analyzed.