The GR binds to an nGRE in the CD95L promoter. (A) The CD95L promoter was searched for potential GR binding sites by comparing the promoter sequence to the consensus of well-characterized nGREs (shaded lines). Candidate sites were analyzed in vitro for GR binding by ABCD assay (“Materials and methods”). Biotinylated DNA fragments of different sizes were incubated with Jurkat whole-cell extract as a source for GR. The GRE from the tyrosine aminotransferase promoter (TAT) served as a positive control; TAT oligo with a mutated GRE (TATmut), as negative control. (B) Overview of the binding elements used. Conserved halfsites are shaded gray; nonconserved halfsites are boxed. The nGRE search pattern is shown in International Union of Pure and Applied Chemistry (IUPAC) nomenclature. (C) Same as in panel A, but using DNA fragments identified as positive for GR binding containing mutations in the conserved base pairs of the putative nGREs. Only 50% of the TAT sample was loaded here compared with the other samples.