After treatment, cell cultures were transferred to Eppendorf tubes and harvested by centrifugation at 2,000 rpm for 5 min. Cells were washed twice with cold PBS and then lysed in 400 µL RIPA buffer, 5 µL protease inhibitor, and 5 µL EDTA for 5 min. Lysed cells were sonicated for 1 min on ice, centrifuged for 10 min at 4°C and the supernatant was transferred into new vials. Equivalent amounts of total protein from each sample were used for SDS-PAGE Western blot analysis. The housekeeping genes β-actin and β-tubulin were used as loading controls. Rabbit anti-rat GH, anti-rat LH, and anti-mouse PRL antibodies were obtained from the National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program. Rabbit anti-human GHRHR antibodies were purchased from Abcam (Cambridge, MA). Goat anti-rabbit antibodies conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA) were used in our chemiluminescence assay. This experiment was repeated twice.