Pulsatile native GnRH induces SET protein down-regulation in LβT2 cells.

<p>(a) LβT2 gonadotrope cells were treated with GnRHa (100 nM) for 2 hours and SET protein (<i>left panel</i>) and SET mRNA (<i>right panel</i>) levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (SET mRNA) and are expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by t test for unpaired groups *: p<0.05, compared to no GnRHa. (b) LβT2 gonadotrope cells were cultured in perifusion chambers as described in “Materials and methods” and challenged or not with pulsatile GnRH (10 nM) at high and low frequencies (one pulse every 0.5 hour or one pulse every 2 hours, respectively). At the end of the incubation, proteins and mRNA were extracted and SET protein and <i>Set</i>, <i>Lhb and Fshb</i> transcripts levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (<i>Set</i>, <i>Lhb and Fshb</i> mRNA) and expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. One-way ANOVA followed by Tukey’s test, ***: p<0.001, compared to no GnRH.