As the fold transform versus saline-treated animals. Enrichment is calculated as the IP versus input ratio and shows the abundance on the transcript within the targeted cell type (IP) when compared to equal amounts of RNA in the whole testis (input). Sequences from the distinct primer sets utilized have been obtained from KPT-8602 Primerbank (http://pga.mgh. harvard.edu/primerbank/) [24] and may be located in Table S6.Immunoprecipitation AssaysAfter therapies, testes were homogenized and immunoprecipitation was performed as described previously [20] with minor modifications. Briefly, ten ul of anti-HA antibody (Covance) had been coupled to 200 ul of beads in citrate-phosphate buffer pH five.0 and also the antibody-bead complicated was added towards the cleared homogenates and incubated overnight at 4uC. Just after incubation, beads have been washed in high salt buffer three occasions for five min, resuspended in RLT buffer (with beta-mercaptoethanol; Qiagen) and stored at 280uC until RNA extraction.LH, FSH and Testosterone Serum DeterminationAfter treatments, blood was obtained by cardiac puncture and permitted to clot in Microtainer serum separator tubes (BectonDickinson) for 1 h at RT. Serum was recovered by centrifugation and stored at 280uC for later evaluation. LH and FSH serum levels were determined by RIA in the University of Virginia Center for Investigation in Reproduction Ligand Assay and Analysis Core, and testosterone levels were determined working with the Testosterone EIA kit (Cayman Chemical).MA-10 Cell CultureCells were cultured as described in [25]. 16105 cells/ml had been seeded onto gelatin-coated 6-well plates and permitted to attach for 2 days. For LH treatment, cells had been incubated in serum-free medium containing 1 BSA overnight before the addition of purified human LH (0.2 Units/ml). Cells have been lysed in SDS sample buffer (62.5 mM Tris, pH six.8, two SDS, 10 glycerol) for protein evaluation, in RLT buffer (Qiagen) for RNA extraction, or as described in [26] for polysome analysis. Sucrose density gradient fractionation and polysome evaluation was performed as described in [20].RNA ExtractionRNA from the inputs (50 ul) and the pellets was obtained utilizing the RNeasy mini kit (Qiagen) based on manufacturer’s directions. Total RNA was quantified utilizing a NanoDrop 1000 spectrophotometer (Thermo Scientific) and its good quality was assessed applying an Agilent 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies).Western Blot AnalysisProtein lysates were quantified utilizing the BCA protein assay (Pierce). Right after protein determination, 20 ug of protein have been separated by SDS-PAGE and transferred onto nitrocellulosePLOS One particular | www.plosone.orgRegulation of Sertoli and Leydig Cell Transcriptsmembranes. Right after transfer, membranes had been blocked with 5 milk in TBST (Tris-buffered saline containing 0.1 Tween-20) and incubated overnight with the following major antibodies: anti-Aquaporin two (1:1000; Novus), anti-phospho-S6 (1:2000; Cell Signaling Technologies) and anti-Beta Actin (1:50,000; SigmaAldrich). Soon after incubation with secondary antibodies (1:10,000; Jackson ImmunoResearch), membranes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20356390 were washed in TBST and developed using an enhanced chemiluminescence (ECL) detection system (Pierce).Immunohistochemistry (IHC)Mice have been perfused transcardially with Bouin fixative or PBS containing four paraformaldehyde and testes were removed, postfixed overnight and cryoprotected with 30 sucrose in PBS. For cryosectioning, testes had been frozen for five min in dry ice and sectioned applying a freezing microtome (Leica). Frozen sections we.
