Figure 3

Linker scanning mutagenesis of the -450 to -351 region of the LAT promoter. Sequential 10 bp mutations were introduced throughout the -450 to -351 region in the context of the -1000 to -1 promoter fragment and the effects on promoter activity were determined in Jurkat T cells. A. The wild-type promoter sequence from -450 to -351 is shown on top. The lower strand depicts each 10 bp change, which incorporates a Not I restriction site (GCGGCCGC) plus one flanking nucleotide on each side. Base pair numbering is according to the translation start site, which was designated +1. B. Promoter activity for each linker scanning mutant was determined following transient transfection into Jurkat T cells. Following normalization of each sample based on the activity of co-transfected control pRL-TK plasmid encoding Renilla luciferase, the fold activation relative to empty pGL3-basic was calculated. The graph depicts the average fold activation and s.e.m. from three or more independent experiments.