Membranes have been washed with TTBS four occasions for five min each, incubated with a 1,2000 dilu tion of anti rabbit horseradish peroxidase antibody for 1 h. The immunoreactive bands have been detected by ECL reagents. Total RNA extraction and gene expression For reverse transcription PCR analysis, total RNA was extracted from mouse brain endothelial cells stimulated by ET 1, as previously described. The cDNA obtained from 0. five ug total RNA was utilised as a template for PCR amplification. Oligonucleotide primers had been created according to Genbank entries for mouse COX 2 and B actin. The following primers had been used for amplification reaction, for. PCR mixes contained 10 ul of 5X PCR buffer, 1. 25 mM of each dNTP, one hundred pmol of each and every forward and reverse primer, and two. five units of Taq polymerase. The final reaction volume was 50 ul.
Amplification was performed in 25 cycles at 94 C, 20 s, 60 C, 40 s, 72 C, 40 s. Just after the last cycle, all samples were incubated for an more 10 min at 72 C. PCR fragments had been analyzed on 2% agarose 1X TAE gel containing ethidium bromide and their size was when compared with a molecular weight marker. Amplification inhibitor OTX015 of B actin, a comparatively invariant internal reference RNA, was performed in parallel, and cDNA amounts had been stan dardized to equivalent B actin mRNA levels. These primer sets especially recognized only the genes of interest as indicated by amplification of a single band of the expected size and direct sequence evaluation in the PCR products. Immunofluorescence staining Cells have been plated on 6 well culture plates with coverslips.
Cells had been shifted to a serum totally free DMEM F 12 for 24 h and treated with ten nM ET 1. Right after washing twice with ice cold PBS, the cells had been fixed with 4% parafor maldehyde in PBS for 30 min, then permeabilized Pim inhibitor with 0. 3% Triton X 100 in PBS for 15 min. The staining was performed by incubating with 10% typical goat serum in PBS for 30 min followed by incubating having a key anti p65 NFB polyclonal antibody for 1 h in PBS with 1% BSA, washing thrice with PBS, incubat ing for 1 h with fluorescein isothiocyanate conju gated goat anti rabbit antibody in PBS with 1% BSA, washing thrice with PBS, and ultimately mount ing with aqueous mounting medium. The images observed beneath a fluorescence microscope. Chromatin immunoprecipitation assay To detect the in vivo association of nuclear proteins with mouse COX 2 promoter, chromatin immunoprecipitation analysis was carried out as previously described.
Briefly, the bEnd. 3 cells had been cross linked with 1% formalde hyde for ten min at 37 C and washed thrice with ice cold PBS containing 1 mM phenylmethylsulfonyl fluoride and 1% aprotinin. Soluble chromatin was prepared applying a ChIP assay kit according to the manufac turers suggestions and immunoprecipitated without having or with anti p65 NFB antibody and normal goat immunoglobulin G.