Following washes and elution, precipitates were heated overnight at 65 C to reverse cross linking of DNA and protein. DNA fragments were purified by phenol chloroform extraction and ethanol precipitation. The purified DNA was subjected to PCR amplification utilizing the primers specific for the region containing the NFB binding web page present inside the COX two promoter re gion, sense primer, PCR frag ments had been analyzed on 2% agarose in 1X TAE gel con taining ethidium bromide plus the size was when compared with a molecular weight marker. Plasmid construction, transient transfection and luciferase assays The mouse COX 2 promoter was constructed as described previously with some modifications. The upstream region of the mouse COX two pro moter was cloned for the pGL3 simple vector containing the luciferase reporter method.
Introduction of a double point mutation into nvp-auy922 molecular weight the NFB binding site to generate pGL COX2 m?B was per formed employing the following primer. The underlined nucleotides indicate the positions of substituted bases. The mutant construct was cloned into the pGL3 basic vector containing the luciferase reporter method. All plasmids had been prepared by using QIAGEN plasmid DNA preparation kits. The siRNAs for p42, p38, JNK1, p65, and scrambled control have been from Dharmacon Study Inc, and NFB or COX two pro moter constructs have been transfected into cells utilizing the Lipofetamine 2000 transfection reagent as outlined by the instructions of manufacture. The transfection efficiency was determined by transfection with enhanced EGFP. To assess promoter activity, cells have been collected and disrupted by sonication in lysis buffer.
After cen trifugation, aliquots on the supernatants were tested for luciferase activity utilizing a luciferase assay system. Firefly luciferase activities had been standardized to B galactosidase activity. Measurement of PGE2 release The cells had been seeded in 12 properly plates and grown to con fluence. Cells were selleck inhibitor shifted to serum absolutely free DMEM F 12 medium for 24 h, after which treated with ET 1 for a variety of time intervals. The culture supernatants have been collected to measure PGE2 levels using an EIA kit as specified by the manufacturer. Statistical analysis of information All information have been estimated using GraphPad Prism Program. Quantitative information had been ana lyzed by 1 way ANOVA followed by Tukeys honestly considerable distinction tests involving person groups. Data had been expressed as imply SEM. A value of P 0. 05 was regarded significant. Background Protein kinase D constitutes a novel family of diacylglycerol responsive serine threonine pro tein kinases with various structural, enzymological and regulatory properties in the protein kinase C family members.