, 2008) with minor modifications Prior to bleaching, neurons wer

, 2008) with minor modifications. Prior to bleaching, neurons were imaged every 30 s for 2 min at 15% laser power. For photobleaching, ROI was exposed to 75% laser power every 1.6 s for 40 frames. Recovery was monitored every 60 s

over 20 min at 15% laser power. To test for translation dependence, we pretreated cultures with 50 μM anisomycin for 30 min before the photobleaching BMN 673 sequence. FRAP quantification and statistical tests are detailed in Supplemental Experimental Procedures. Dissociated DRG cultures were transfected with Dendra2 reporter constructs fused to Importin β1 3′ UTR axonal and cell body variants using Amaxa nucleofection. Dendra2 was photoconverted using a 405 nm laser at 5% energy power and 40× oil objective for 30 s. Images were captured every 4 min under the AT13387 in vitro same conditions using 488 nm (0.1% energy) and 559 nm (4%

energy) lasers. The proximal region was photoconverted using a 405 laser at 0.5% energy power for 2 min every 4 min. Dendra2 quantification and statistical tests are detailed in Supplemental Experimental Procedures. L4/L5 DRGs were dissected from crush-lesioned or control animals at the indicated time points. Total RNA pooled from three animals was extracted using Trizol (TRI, Sigma-Aldrich). Total RNA (200 ng) was amplified, labeled, and hybridized on Illumina arrays (MouseRef-8 version 2.0 Expression BeadChip Kit). Data analysis was performed in the R environment using Bioconductor packages (http://www.bioconductor.org). Briefly, log2-transformed data was normalized using quantile normalization and differential expression analysis was performed using the LIMMA package as previously described PAK6 (Coppola, 2011). Total RNA was extracted from total DRGs pooled from three adult animals per replicate, using the Trizol reagent (TRI, Sigma-Aldrich) according to manufacturer’s instructions. Replicates consisting of at least 10 μg of total RNA each were processed for RNA expression analysis (RNA-Seq) on an Illumina Genome Analyzer IIx at the

High-Throughput Sequencing Unit in the Weizmann Institute of Science. RNA-Seq data was analyzed using DESeq (Anders and Huber, 2010). CatWalk training was carried out as previously described (Deumens et al., 2007). Motivation was achieved by a combination of food restriction during the initial training and placing of palatable reward at runway ends. Data were collected and analyzed with CatWalk software version 9.0 at days 0, 2, 4, 6, 8, 10, 14, 18, 22, and 26 postinjury. The analyzed indices are shown as a ratio between the ipsilateral (right) hind paw and contralateral (left) hind paw and are expressed as mean ± SEM. Quantification and statistical tests are detailed in Supplemental Experimental Procedures. We thank Erin Schuman for the myristylated GFP reporter, Freda Miller for the Tα1 tubulin promoter, and Fan Wang for the Advilin-Cre mice.

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