, 2011; Dieterich et al., 2008; Lai et al., 2008; Thompson et al., 2004), in synapse to nucleus signaling during development ( Higashi-Kovtun et al., 2010; Mosca and Schwarz, 2010; Ting et al., 2007), and even in cytoplasmic transport of nucleic acid cargos in nonneuronal check details cells ( Mesika et al., 2005; Salman et al., 2005). The mechanisms and models established in this study should be useful in future scrutiny of these additional specific roles of importins. Finally, application of similar subcellular

targeting approaches to other transcripts in distal cytoplasm will help to determine the scope of contribution of mRNA transport and translation mechanisms to spatial discrimination in cellular functions. Adult (8–12 weeks old) male Wistar rats were from Harlan Laboratories. All mouse strains were bred and maintained at the Veterinary Resources Department of the Weizmann Institute. Sciatic nerve crush and DRG culture preparations were as previously described (Hanz et al., 2003). GFP transgenic mice were generated as previously described (Willis

et al., 2011) with modifications as detailed in Supplemental Experimental Procedures. A conditional Cre/lox-mediated knockout in the 3′ UTR region of Importin β1 was generated by flanking the targeted 1.1 kb sequence segment with loxP sites to allow for Cre-mediated deletion. Three SV40 polyadenylation signals were inserted immediately downstream of the floxed region in the constructs to ensure stability of the truncated

mRNA. For further details, please see Supplemental Experimental selleckchem Procedures. QPCR was performed as described (Nilsson et al., 2005) using Taqman primer kits for β-actin (normalization control), GFP, and Importin β1. Immunostaining was carried out as previously described (Hanz et al., 2003; Perlson et al., 2005); for antibody details, please see Supplemental Experimental Procedures. Antisense oligonucleotide probes for Importin β1 were designed using Oligo 6 software and checked for homology and specificity by BLAST. To Edoxaban detect GFP mRNA in transfected DRGs, we generated antisense and sense GFP riboprobes by in vitro transcription using digoxigenin-labeled nucleotide mixture (Roche). Hybridization was performed at 55°C for 4 hr and then samples were processed for immunodetection with Cy3-conjugated mouse anti-digoxigenin (1:200; Jackson Immunoresearch). Hybridization to tissue sections was performed as previously published ( Muddashetty et al., 2007), with minor modifications as detailed in Supplemental Experimental Procedures. Isolation of DRG axons was carried out as previously described (Zheng et al., 2001). Two hundred nanograms of RNA from cell body and axons were used as a template for reverse transcription and PCR. For details of reaction conditions and primers, please see Supplemental Experimental Procedures. Dissociated DRG cultures were transfected with reporter constructs fused to Importin β1 3′ UTR axonal and cell body variants using Amaxa nucleofection.