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. Author manuscript; available in PMC: 2010 Sep 17.
Published in final edited form as: Mol Endocrinol. 2006 May 4;20(9):2062–2079. doi: 10.1210/me.2005-0316

Fig. 1. Androgens, Progestins, and Glucocorticoids Induce FSHβ Gene Expression in Immortalized Gonadotropes.

Fig. 1

A, RT-PCR was used to detect expression of the steroid receptors in LβT2 cells. Amplified AR is visible at 550 bp, PR at 406 bp, and GR at 298 bp. In reverse transcription of total RNA isolated from LβT2 cells, + indicates the presence of reverse transcriptase, and − indicates no reverse transcriptase. B, ChIP was performed using cross-linked protein/chromatin from LβT2 cells and antibodies directed against AR, PR, and GR or, as a negative control, against nonspecific IgG. Upper panel, PCR primers encompassing the proximal promoter of FSHβ were used to detect precipitation of genomic DNA. Lower panel, PCR primers encompassing the downstream FSHβ coding region were used as a control for specificity. PCR amplification was performed on 0.2% chromatin input (lane 1), and chromatin was precipitated with either mouse IgG (lane 2), AR (lane 3), PR (lane 4), or GR (lane 5) antibodies. C, The −1000FSHβluc reporter gene was transiently transfected into LβT2 cells along with 200 ng of the respective steroid receptor expression vectors. After overnight starvation in serum-free media, the cells were treated with 100 nm testosterone, DHT, progesterone, corticosterone, or 17β-estradiol for 24 h. Luciferase activity was normalized to β-galactosidase activity and set relative to the empty reporter vector. The results represent the mean ± sem of at least three experiments performed in triplicate and are presented as fold induction of hormone treatment relative to the vehicle control (ethanol). For cells transfected with AR, and treated with testosterone or DHT, * indicates significantly different from the vehicle-treated control, using one-way ANOVA followed by Tukey’s post hoc test. For LβT2 cells transiently transfected with PR, GR, ERα, or ERβ and treated for 24 h with progesterone, corticosterone, or 17β-estradiol, respectively, * indicates significantly different from the respective vehicle-treated control using Student’s t test. RT, Reverse transcriptase.