, 2005 and Scherer et al., 2005). This construct should direct the expression of inactive RafTR protein in these cells throughout the PNS. The RafTR fusion protein contains a point mutation in the human estrogen receptor ligand-binding domain, so that it can be activated by the injection of the estrogen analog tamoxifen but not by endogenous estrogens. Following pronuclear injection of the linearized construct, we obtained 8 RafTR-positive mice, Navitoclax chemical structure two of which produced lines with detectable expression of RafTR mRNA and protein in sciatic nerve. The RafTR-expressing mice were crossed with wild-type (WT) animals, and in all subsequent experiments heterozygous P0-RafTR mice

were compared with their WT, age-matched littermates. Similar results were obtained with both P0-RafTR

lines. buy Bortezomib To confirm the specificity of expression of the construct and the inducibility of the Raf-kinase activity, we initially gave the mice a single intraperitoneal (IP) injection of tamoxifen and analyzed the mice after 24 hr. As controls, we compared the injected P0-RafTR mice to both uninjected P0-RafTR mice and to tamoxifen-injected WT controls. Western blot analysis detected the RafTR protein in peripheral nerve extracts from P0-RafTR mice but not in WT controls (Figure 1B). Moreover, the fusion protein could not be detected in cortical brain extracts, confirming the specificity of expression. Levels of RafTR protein were higher in the injected animals compared to uninjected RafTR-expressing controls, which is consistent with the reported stabilization of the protein upon tamoxifen binding. Importantly, Raf-kinase activity, as measured by the level of the phosphorylated downstream effector ERK (P-ERK), was induced in the PNS but not the CNS following tamoxifen injection (Figure 1B). Increased levels of P-ERK were not detectable in either WT animals injected with tamoxifen or uninjected P0-RafTR animals, confirming the tight

regulation of the Raf kinase in the mouse. Immunolabeling of sciatic nerves demonstrated that ERK was activated specifically in myelinated Schwann cells following tamoxifen injection, confirming the inducible nature of the kinase in the intended heptaminol target cells (Figure 1C). The magnitude of P-ERK induction was similar to that seen in the distal stump of cut WT sciatic nerves 24 hr following nerve transection, indicating that we are activating the ERK pathway in P0-RafTR nerves to levels similar to those seen following an injury response in WT nerves (Figures 1B, 1C, and S1B). Following injury, high levels of P-ERK are rapidly induced in myelinating Schwann cells and persist for 3–5 days (Harrisingh et al., 2004). To mimic this, control and transgenic mice (aged 4–6 weeks) were given 5 consecutive daily IP tamoxifen injections and their behavior was monitored daily.