Localization of dominant-negative ETS protein in human endothelial cells. (A) Structure of the dominant-negative peptide A1 used to block ELF-1 function, including a biotinylated amino terminus, the terminal portion of the ETS domain corresponding to ETS peptide A, and a 12–amino acid HIV-TAT protein membrane–transducing domain (see “Materials and methods” for the sequence). (B) Time-dependent entry of the dominant-negative ETS peptide into the nucleus of HUVECs at different time points (0.5, 1, and 6 hours) is shown. Cells were fixed at various time points and immunostaining was performed to detect the biotinylated peptide. Nuclear localization was performed using the DAPI stain, and evidence of colocalization was demonstrated by merging the 2 images. Images were taken at × 20 magnification for 0.5-, 1-, and 6-hour time points, and also at × 40 for the 6-hour time point (bottom panel).