electrophoretically separated on 10–15% discontinuous acrylam- ide gels, transferred to pure nitrocellulose membranes (PRO- TRAN BA85, Schleicher and Schuell, 0.45 mm), and the equal loading and transfer of the samples was monitored by Ponceau staining of the membranes. The different proteins were visualized by incubation with specific antibodies overnight at 4uC, followed by incubation with a secondary antibody for two hours at room temperature. The rabbit polyclonal antibody against RXR-c (Anaspec) was used at a dilution of 1:1,000. Secondary horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoRe- search) were used at a dilution of 1:10,000. The blots were developed by chemiluminescence (Immun-StarTM HRP Chemi- luminiscent Kit, Bio-Rad) as indicated by the manufacturer. Blots were quantitated with the imaging and analysis software ImageJ 1.42d (http://rsbweb.nih.gov/ij/). Real time quantitative PCR RNA was extracted using TRIH Reagent (Sigma) and quantitated with a NanodropTM spectrophotometer (Thermo Scientific). 1 mg total RNA from each sample was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad) and the relative levels of the genes of interest determined by quantitative real time PCR using FastStart Universal SYBR Green Master (ROX) mix (Roche) in a Mx3005P instrument (Stratagene). Every measure was always performed in duplicate (two PCR reactions from the same RT sample) and the average of those technical replicates was considered as the Ct for that particular sample. All gene expression levels were normalized to the housekeeping genes 18S rRNA or cyclophilin A. The primers were designed by using the MacVector suite (http://www.macvector.com/) coupled with Amplify software (http://engels.genetics.wisc.edu/amplify/). Primers were designed from common sequences in the rat and mouse genes and, when possible (multiexonic genes), spanning two consecutives exons to avoid interference from possible genomic DNA contamination. For monoexonic genes, a DNase treatment of the samples was performed prior to cDNA sysnthesis. In all cases, primers were first tested by conventional RT-PCR to produce a single amplicon of the correct size, followed by Q-RT-PCR to avoid primer-dimer artifacts and ensure a lineal range of quantitation. When several mRNA splice variants were present for a given gene, the primers Regulation of RAR-b and RXR-c in Schwann Cells were always designed to recognize all known isoforms. The primers used are indicated in Supp. Info. Table S1. Determination of hnRNA hnRNA levels were also determined by RT-Q-PCR, with the following differences from regular mRNA determination. The samples were always treated with DNase to prevent genomic DNA contamination and reverse transcribed with iScript Select cDNA Synthesis Kit (Bio-Rad) using specific oligonucleotides for the genes of interest as primers (the same reverse primer used in the subsequent PCR). hnRNA levels were determined by using primers from an exon and a contiguous intron (exon 3 and intron III for RAR-b and exon 2 and intron II for RXR-c), so DNA amplification only could occur from newly formed, immature non- spliced RNA. Statistical analysis Throughout the whole article, data shown is representative of at least two separate experiments performed each time at least in duplicate (experimental replicates). Significance was determined by Student’s t-test (unpaired) analysis using StatPlus:mac LE2009 coupled with Microsoft Excel:mac 2008. Unless indicated otherwise, all comparisons were made against their respective controls. Throughout the test, differences were considered as significant when p,0.05. In the Figures, three different levels of significance (p,0.05; p,0.01 and p,0.005) were considered. Results Axonal contact up-regulates RXR-c in SCs We have recently shown that retinoic acid regulates myelin formation in the peripheral nervous system. In order to analyze which receptors were present in SCs, we performed real time RT- Q-PCR from primary Schwann cell cultures compared to primary DRG sensory neurons and total brain. In general, with the exception of RAR-b, all retinoid receptors, RARs and RXRs, were expressed by isolated Schwann cells at levels comparable or superior to DRG sensory neurons or total brain, which is known to express all retinoid receptors at relatively high levels (Fig. 1A). Interestingly, not all retinoid receptors were homogenously maintained during the different stages of myelin formation or Figure 1. RXR-c expression is increased in DRGN/SC cocultures. (A) The mRNA levels of all six RARs and RXRs were analyzed by Q-RT-PCR. The results are shown as the mean value 6 SD relative to the levels found in whole adult rat brain (dashed line). (B) The mRNA levels of RAR-b and RXR-c were analyzed by Q-RT-PCR in isolated SCs or myelination-competent DRGN/SC cocultures and compared to isolated DRG neurons and total adult rat brain. The results are shown as the mean value 6 SEM relative to the levels found in isolated SCs. doi:10.1371/journal.pone.0017023.g001 PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17023