GnRH-(1–5) Receptor Signaling in GN11 Cells

GnRH-(1–5) was purchased from Bachem (Torrance, CA, USA) and reconstituted to 10 mM in distilled water and stored in 12 µL aliquots at −80°C. GN11 cells (18 (link)) generously donated by Dr. Sally Radovick (Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech Inc., Herndon, VA, USA) without antibiotics and supplemented with 7% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 3% newborn calf serum (Hyclone), 25 mM glucose, and 5 mM l-glutamine (7 (link), 11 (link), 19 (link)). Cells were maintained at 37°C in an atmosphere with 5% CO₂. In addition, all experiments were conducted at 37°C in an atmosphere with 5% CO₂. All GN11 cells used for experimentation were below passage 25. GN11 cells originated from an olfactory tumor extracted from a male mouse (18 (link)). The antibodies used in this study were specific for the TGF-βRI (1:1,000; Cat. No. SC-9048, Santa Cruz Biotechnology, Dallas, TX, USA), TGF-βRII (1:1,000; Cat. No. SC-400, Santa Cruz Biotechnology), GAPDH (1:2,000; Cat. No. SC-25778, Santa Cruz Biotechnology), and anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000; Cat. No. 170-6515, Bio-Rad, Hercules, CA, USA).