Figure 3. Exogenous prolactin activates FASN gene promoter activity by engaging PRLR.Top. Estradiol-depleted cells were transiently transfected with a plasmid containing a luciferase gene driven by a 178-bp FASN gene promoter fragment harboring a SREBP-binding site, flanked by auxiliary NF-Y and Sp-1 sites as described in Figure 2C, 2D. The next day, cells were treated with 200 ng/mL prolactin (PRL) in the absence or presence of a 5-fold-excess of the prolactin antagonist hPRL-G129R (1000 ng/mL) in 0.5% CCS. After ~24 h of incubation, cells were lysed, luciferase activity was measured and relative (fold) changes in transcriptional activities of FASN promoter-luciferase-transfected cells were calculated. The data are shown as the means (columns) ± S.D. (bars) from three separate experiments (performed in duplicate). Luciferase activity in prolactin- and/or hPRL-G129R-treated cells was compared with that in vehicle-treated control cells (* P < 0.05; ** P < 0.005). Bottom. Estradiol-depleted cells were treated with 200 ng/mL PRL in the absence or presence of a 5-fold excess of hPRL-G129R in 0.5% CCS for 48 h. Immunoreactive bands for PR-A, PR-B, and PRLR proteins were analyzed by immunoblotting as described in Figure 2A. β-actin was used to control for protein loading and transfer. Figure shows a representative immunoblot analysis. Similar results were obtained in 3 independent experiments.