<p>(a) Total RNA isolated from whole brain from <i>Crh+/+</i> mice (lane 1), NMF isolated from <i>Crh+/+</i> mice (lane 2) and NMF isolated from <i>Crh−/−</i> mice (lane 3) was subjected to RT-PCR for evaluation of <i>Crh</i> expression. Lane 4 represents a sample without RT-enzyme and lane 5 represents a sample without template. (b) <i>Crf1</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from whole brain, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (c) <i>Crf2</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from heart, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (d) Effects of antalarmin and astressin-2B on specific [<sup>125</sup>I] Tyr0-sauvagine binding to CRF<sub>1</sub> and CRF<sub>2</sub>. Membrane homogenates from <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts were assayed for specific binding with [<sup>125</sup>I] Tyr0-sauvagine, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a>. The bars represent the % decrease of specific binding. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. (e) Effect of CRH on cAMP accumulation in <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts. Stimulation of cAMP accumulation by CRH was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a> in intact cells. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. * represents statistical difference (P<0.05) between genotypes exposed to the same treatment and # represents statistical difference (P<0.05) between different treatments in the same genotype.</p
