Figure 3.

Inhibition of PKA Activity Markedly Attenuated GnRH Stimulation of FSHβ Promoter Activity with Only Modest Effects on LHβ Promoter Activity. A, LβT2 cells in static culture were transiently transfected with 2 μg −140/+15 FSHβLUC and pretreated 40 hours after transfection for 30 min with the indicated concentrations of H89, followed by cotreatment with vehicle or 10 nM GnRH for 4 hours. B, LβT2 cells in static culture were transiently transfected with 2 μg of −140/+15 FSHβLUC, as well as 2 μg empty vector (EV, black solid bars); 1 μg empty vector in combination with 1 μg DNPKA (EV:DN, gray shaded bars); or 2 μg DNPKA alone (DN, white solid bars). C, LβT2 cells were transiently transfected with 2 μg of −797/+5 LHβLUC as well as the EV, EV:DN, and DN vectors as described in panel B. Panels B and C were treated with vehicle or 10 nM GnRH for 4 hours. Bar graphs show relative luciferase activity (mean ± SEM from three independent experiments each performed in triplicate, normalized to β-galactosidase activity). Significant differences (P < .05), measured by one-way ANOVA with a post hoc Tukey multiple-comparison test, are indicated by different letters.