Fig. 5. Mutation of the HD Element Abrogates LβT2 Cell Specificity.
Site-directed mutagenesis was performed to mutate the 5′ Egr-1 binding site, the 3′ SF-1 binding site, the 5′ SF-1 binding site, and the HD in both the −1800LHβLuc and −122LHβLuc reporter genes. Two different HD mutations were examined (m4 and m5) as indicated in the diagram at left. HDm4 and HDm5 correspond to mutant 4 and mutant 5 used in Figs. 4 and 8 and shown in Table 1. Transient transfections of mutant LHβLuc plasmids were performed in LβT2, αT3-1, and NIH3T3 cells (identified by black, gray, and white bars, respectively). A, Mutations in the context of −1800LHβLuc. B, Mutations in the context of −122LHβLuc. Results represent the mean ± SEM of at least three independent experiments, each performed in triplicate (n ≥ 9). Asterisks (*) designate a significant difference from αT3-1 and NIH3T3 cells (P < 0.05); # indicates a significant difference from NIH3T3 cells (P < 0.05).