FIG. 5.
Brd4 and SPA-1 interact in the nucleus. (A) Confocal images of endogenous Brd4 (green) and SPA-1 (red). NIH 3T3 cells (105) grown on a coverslip were stained with Brd4 or SPA-1 antibodies. Merged images show superimposition of green and red signals. DNA was counterstained with Hoechst stain (blue). (B, C, and D) BiFC assay. NIH 3T3 cells (105) seeded on a coverslip were transfected with the combination of plasmids indicated on the left (0.4 μg of each vector) for 24 h. The emission of YFP fluorescence was detected on a confocal microscope. (B) Yellow fluorescence is detectable in the nuclei of NIH 3T3 cells cotransfected with Y(C)-Brd4 and Y(N)-SPA-1 due to complementation of the two YFP fragments. (C) Y(N)-ΔGRDSPA-1, when coexpressed with Y(C)-Brd4, does not show a YFP signal, whereas Y(N)-ΔPDZSPA-1 complements Y(C)-Brd4 and shows a yellow signal. (D) Brd4 mutant Y(C)-ΔC, when coexpressed with Y(N)-SPA-1, generates a YFP signal, whereas Y(C)-ΔI&II does not. The same cells were fixed and stained with Rap2 antibody (red). Merged images show superimposition of yellow and red signal or yellow, red, and blue (Hoechst stain) signal. (E) Rap2 staining and merged images of BiFC-Rap2 or Rap2-Hoechst stain. Cells were transfected with Y(C)-Brd4 and Y(N)-SPA-1 and subsequently stained with Rap2 antibody, to indicate the presence of Rap2 in the nucleus (shown by arrowheads), which partially overlaps the BiFC signal.