Figure 3

Expression of nNOS is reduced in the TR4^-/- penis, and TR4 binds and activates the nNOS promoter. A. Qualitative RT-PCR (upper panel) and real time RT-PCR quantitation (lower panel) of nNOS gene expression in the penis. Expression data were obtained, in triplicate, from each of two TR4^+/+ and two TR4^-/- mice. β-actin levels were determined as a control for template amount in PCR reactions. B. Western blot analysis of penis samples from TR4^+/+ and TR4^-/- mice (with priapism) demonstrates reduced nNOS protein expression in TR4^-/- tissue. Samples from each of 3 TR4^+/+ and 3 TR4^-/- mice were analyzed, and a representative blot is shown. C. Transactivation assay for TR4 regulation of nNOS expression. TR4 can activate the nNOS exon 2 promoter and control basal nNOS transcription. pEx2 (0.2 μg) and increasing amounts of TR4 were cotransfected into CV-1 cells. Promoter activity of each sample was normalized to the average activity of the pEx2 promoter when transfected alone. Results are the mean ± SEM from at least three experiments performed in duplicate. ***P < 0.01 d. By EMSA assay, TR4 is shown to bind to the nNOS-NHR probe (TR4/nNOS-NHR). The presence of TR4 in the complex was confirmed by supershift (TR4/nNOS-NHR/Ab) using a TR4-specific antibody. n.s., nonspecific binding.