Fig. 2

Effect of testosterone on Tac3 gene and NKB protein expression in mHypoA-55 cells. mHypoA-55 cells were stimulated with the indicated concentrations of testosterone for 24 h. A After stimulation, mRNA was extracted and reverse transcribed. Tac3 mRNA levels were measured by quantitative RT-PCR. Samples for each experimental group were run in duplicate and normalized to the mRNA levels of GAPDH as a housekeeping gene. The results are expressed as fold induction over unstimulated cells and presented as the mean ± SEM of three independent experiments. *P < 0.05 vs. control. B After stimulation, lysates (20  g protein) from mHypoA-55 cells were analyzed by SDS-PAGE followed by immunoblotting and incubation with anti-NKB antibodies. The bands were visualized using an HRP-conjugated secondary antibody. C Scanning densitometry of bands was performed using ImageJ to determine differences in kisspeptin protein expression, with normalization to β-actin levels. Results are expressed as fold stimulation over the unstimulated group/control. Values are the mean ± SEM of fold stimulation from independent experiments. **P < 0.01 vs. control