Figure 9

The transcription factor Runx1 binds to the LAT promoter. A. A DNA probe encompassing the Runx site and the Ets site centered at -360 (Runx/-360 Ets wt) was labeled with ³²P and incubated with nuclear extracts from Jurkat T cells in the absence or presence of 100-fold molar excess of unlabeled competitor DNA. Samples were processed as described in the legend for Fig. 5. For competition experiments, the unlabeled competitor DNA was the same sequence as that of the labelled probe (self wt), the same sequence as the probe except containing point mutations within the Ets site (self mutEts), the Runx site (self mutRunx) or both sites (self mutEts/Runx). B. Experiments were conducted as in Fig. 9A, with the exception that the Elf-1 antibody was also added to the indicated sample. C. Experiments were conducted as described in Fig. 9A except that the Runx-1 antibody was added instead of unlabeled competitor. For A-C, the arrow denotes specific complexes. D. Jurkat and RBL-2H3 cells were transfected with the wild type -1000 to -1 reporter plasmid or the -1000 to -1 reporter plasmid with a mutation within the Runx site. Following incubation, samples were harversted, luciferase activities were measured and normalized to Renilla luciferase, and the activity obtained with empty pGL3-basic was set at 1-fold. The results shown are the average fold induction and s.e.m. from three or more independent experiments. p < 0.05 for the mutant reporters relative to wild-type as determined using the two-tailed t test.