As the fold change versus saline-treated animals. Enrichment is calculated because the IP versus input ratio and shows the abundance from the transcript in the targeted cell variety (IP) when in comparison to equal amounts of RNA in the complete testis (input). Sequences on the unique primer sets used were obtained from Primerbank (http://pga.mgh. harvard.edu/primerbank/) [24] and may be identified in Table S6.Immunoprecipitation AssaysAfter Dihydrotanshinone I web 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 complex was added for the cleared homogenates and incubated overnight at 4uC. After incubation, beads have been washed in high salt buffer 3 occasions for 5 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 analysis. LH and FSH serum levels have been determined by RIA at the University of Virginia Center for Investigation in Reproduction Ligand Assay and Analysis Core, and testosterone levels were determined making use of the Testosterone EIA kit (Cayman Chemical).MA-10 Cell CultureCells were cultured as described in [25]. 16105 cells/ml have been seeded onto gelatin-coated 6-well plates and allowed to attach for two days. For LH therapy, cells had been incubated in serum-free medium containing 1 BSA overnight before the addition of purified human LH (0.two Units/ml). Cells had been lysed in SDS sample buffer (62.5 mM Tris, pH six.eight, 2 SDS, 10 glycerol) for protein analysis, in RLT buffer (Qiagen) for RNA extraction, or as described in [26] for polysome evaluation. Sucrose density gradient fractionation and polysome analysis was performed as described in [20].RNA ExtractionRNA in the inputs (50 ul) and the pellets was obtained making use of the RNeasy mini kit (Qiagen) as outlined by manufacturer’s directions. Total RNA was quantified making use of a NanoDrop 1000 spectrophotometer (Thermo Scientific) and its quality was assessed using an Agilent 2100 Bioanalyzer with all the RNA 6000 Nano kit (Agilent Technologies).Western Blot AnalysisProtein lysates had been quantified making use of the BCA protein assay (Pierce). Following protein determination, 20 ug of protein had been separated by SDS-PAGE and transferred onto nitrocellulosePLOS A single | www.plosone.orgRegulation of Sertoli and Leydig Cell Transcriptsmembranes. Just after transfer, membranes had been blocked with five milk in TBST (Tris-buffered saline containing 0.1 Tween-20) and incubated overnight together with the following key antibodies: anti-Aquaporin 2 (1:1000; Novus), anti-phospho-S6 (1:2000; Cell Signaling Technologies) and anti-Beta Actin (1:50,000; SigmaAldrich). Following incubation with secondary antibodies (1:10,000; Jackson ImmunoResearch), membranes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20356390 have been washed in TBST and created using an enhanced chemiluminescence (ECL) detection method (Pierce).Immunohistochemistry (IHC)Mice were 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 utilizing a freezing microtome (Leica). Frozen sections we.
