As the fold adjust versus saline-treated animals. Enrichment is calculated because the IP versus input ratio and shows the abundance from the transcript inside the targeted cell form (IP) when in comparison to equal amounts of RNA in the complete testis (input). Sequences from the different primer sets applied were obtained from Primerbank (http://pga.mgh. harvard.edu/primerbank/) [24] and may be identified in Table S6.Immunoprecipitation AssaysAfter treatments, testes were homogenized and immunoprecipitation was performed as described previously [20] with minor modifications. Briefly, 10 ul of anti-HA antibody (Covance) were coupled to 200 ul of beads in citrate-phosphate buffer pH 5.0 plus the antibody-bead D8-MMAF (hydrochloride) supplier complex was added for the cleared homogenates and incubated overnight at 4uC. Following incubation, beads have been washed in high salt buffer three times 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 allowed 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 were determined by RIA in the University of Virginia Center for Investigation in Reproduction Ligand Assay and Analysis Core, and testosterone levels were determined utilizing the Testosterone EIA kit (Cayman Chemical).MA-10 Cell CultureCells had been cultured as described in [25]. 16105 cells/ml have been seeded onto gelatin-coated 6-well plates and permitted to attach for 2 days. For LH therapy, cells were incubated in serum-free medium containing 1 BSA overnight before the addition of purified human LH (0.two Units/ml). Cells have been lysed in SDS sample buffer (62.five mM Tris, pH 6.8, 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 evaluation was performed as described in [20].RNA ExtractionRNA from the inputs (50 ul) as well as the pellets was obtained applying the RNeasy mini kit (Qiagen) as outlined by manufacturer’s directions. Total RNA was quantified utilizing a NanoDrop 1000 spectrophotometer (Thermo Scientific) and its quality was assessed using an Agilent 2100 Bioanalyzer together with the RNA 6000 Nano kit (Agilent Technologies).Western Blot AnalysisProtein lysates had been quantified applying the BCA protein assay (Pierce). Soon after protein determination, 20 ug of protein had been separated by SDS-PAGE and transferred onto nitrocellulosePLOS One | www.plosone.orgRegulation of Sertoli and Leydig Cell Transcriptsmembranes. Just after transfer, membranes had been blocked with 5 milk in TBST (Tris-buffered saline containing 0.1 Tween-20) and incubated overnight together with the following main antibodies: anti-Aquaporin 2 (1:1000; Novus), anti-phospho-S6 (1:2000; Cell Signaling Technologies) and anti-Beta Actin (1:50,000; SigmaAldrich). After 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 making use of an enhanced chemiluminescence (ECL) detection program (Pierce).Immunohistochemistry (IHC)Mice have been perfused transcardially with Bouin fixative or PBS containing 4 paraformaldehyde and testes have been removed, postfixed overnight and cryoprotected with 30 sucrose in PBS. For cryosectioning, testes were frozen for five min in dry ice and sectioned using a freezing microtome (Leica). Frozen sections we.
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