Metab

Metab. be conserved in other mammals, because the rat and human genes are also selectively expressed in reproductive tissues (19, 25C30). Targeted deletion of the founding member of the cluster, gene and insulin protein are expressed in the testis in a developmentally regulated manner and that is a direct target of RHOX5 and identified specific RHOX5 domains and amino acids required for induction. As part of this analysis, we SW-100 identified other RHOX family members that share with RHOX5 the ability to induce and decided the specific domains and homeodomain residues dictating this ability. We showed that this is a conserved response, and we showed that is expressed. Last, we showed that regulates several other metabolism genes, including those that either promote or antagonize insulin action. Together, our analysis suggests that vector (Stratagene, La Jolla, CA). Expression vectors encoding mouse and human RHOX factors have been described previously (37). The 5-flanking sequences in the promoter construct SW-100 (promoter and gene constructs was performed as described previously (40). The polyclonal rabbit antisera against RHOX peptides were kindly provided by IMGENEX (San Diego, CA). Insulin signaling was assessed by Western blot analysis using the Cell Signaling Technology Phospho-Akt antibody sampler kit (catalog no. 9916), GSK3 27C10 (catalog no. 9315), and PTEN (phosphatase and tensin SW-100 homolog) (catalog no. 9552) under the recommended conditions. Analysis was performed on total testes lysate from four individual WT or was used as filler to normalize DNA mass) using Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol. Cells were harvested 36 h post-transfection to measure them for luciferase activity using the Dual-Luciferase Assay (Promega, Madison, WI), following the manufacturer’s instructions. For protein expression vectors, equivalent transfection efficiency and plasmid activity were monitored by expression of GFP from an internal ribosome entry site polycistronic message downstream of the protein expression cassette (37). The data presented SW-100 in the figures are the mean S.E. of at least three independent transfection experiments. Data from transient transfection assays were statistically analyzed by analysis of variance, and differences between individual means were tested by a Tukey multiple-range test using Prism version 4.0 (GraphPad Software). Differences SW-100 were defined as significant if the value was less than 0.05. We purified Sertoli cells and Leydig cells from P12 testes using a protocol involving sequential trypsin, collagenase, and hyaluronidase digestion, followed by hypotonic shock and differential gravity sedimentation (19, 43). The purity of cell fractions was assessed by quantitative polymerase chain reaction (qPCR) analysis for established Leydig, Sertoli, and germ cell markers. Somatic cell preparations that exhibited significant contamination with germ cell markers were discarded and excluded from the analysis. RNA Purification, qPCR, and EMSA Total cellular RNA was isolated as described Rabbit polyclonal to ACOT1 previously (19, 41). Reverse transcription-PCR analysis was performed by first generating cDNA from 1 g of total cellular RNA using iSCRIPT (Bio-Rad) and performing qPCR analysis using SYBR Green incorporation and the method (with ribosomal L19 for normalization), as described previously (19). For EMSA, 32P-labeled blunt-end double-stranded probes (5 104 cpm) were incubated for 30 min at room temperature in 20 l of binding buffer (10 mm Tris (pH 7.9), 50 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol, and 1 g of poly(dI:dC)) containing 2 g of mouse testes extract prepared as described previously (42). For antibody supershift and blocking assays, the reaction mixtures were preincubated with polyclonal antisera at room temperature for 20 min before the addition of warm probe. The DNA-protein complexes were resolved in 4.5C5% nondenaturing polyacrylamide gels at 150 V for 3C4 h at 4 C. Microarray Analysis Total cellular RNA prepared from the testes of six P12 mice (three expression construct (RHOX5, R-177). After 36 h, the cells were incubated with 1% formaldehyde for 10 min at room temperature, followed by incubation with 0.1 m glycine for 5 min at 37 C to stop the fixation. The cells were then washed three times with cold PBS containing 1 mm PMSF and 1% protease inhibitor mixture (Sigma Inc.), harvested, and lysed with cell lysis buffer (5 mm PIPES (pH 8.0), 85 mm KCl, and 0.5% Nonidet P-40) at 4 C for 10 min. The debris was pelleted, and the supernatant was resuspended in.