3 forE DMSO, celecoxib selenocoxib, 2 selenocoxib 3 for 12 h, followed by E. coli LPS stimulation for 2 8 h Sch Estimation of protein in the lysates was rtetest using the H Biocinchoninic and equal amounts of protein for SDS-PAGE and Western blot with specific antibody rpern GPX1, COX-2 and iNOS were used. The density of the protein bands were quantified and normalized to GAPDH. All experiments were repeated at least four times, and a representative Western blot shown in each case. Suppression of DNA-PK pro inflammatory genes by coxibs were also in cultures of primary Macrophages Ren compared derived from mouse bone marrow. BMDMs were from M Nozzles isolated and cultured with C57 BL6 0.1 or 1 M of celecoxib, 2 or 3 for selenocoxib selenocoxib 12 h after stimulation with LPS, followed as described above. Macrophages were cultured in DMEM medium f defining 5 Fetal bovine serum, 2 mM L glutamine, and 1X penicillin streptomycin. BMDMs in the above media with L929 fibroblast conditioned medium were cultivated as a source of M CSF. 2.5. Effect on selenocoxibs PGE2, TXB2 and TNF RAW264.7 cells were treated with 0.1 or 1 M of celecoxib, 2 or 3 selenocoxib selenocoxib DMSO for 12 h prior to the pre-LPS stimulation for 12 hours. SC 560 treated cells were used to prevent a COX-mediated production of PG. The release of PGE2 and TXA2 survived in cell culture media were measured using ELISA kits TXB2 and PGE2. To investigate the anti-inflammatory effect of these compounds on TNF and COX-2, we used real-time PCR. RAW264.7 cells were treated with LPS for 2 h, and total RNA was stimulated using Trizol reagent.
The first strand cDNA was synthesized using the cDNA Archive Kit and in real tests Ispinesib with real-time PCR Taqman probes pre-validated murine TNF, COX-2 and GAPDH. Delta CT values were calculated. 2.6. Preparation of nucleic Ren extracts for testing electrophoretic mobility shift were nucleic Re proteins Isolated as described above. The DNA sequence of the sense strand of doppelstr-Dependent oligonucleotide was specific NF B 5 ? GATCCAGTTGAGGGGACTT TCCCAGGC third Preparations by oligonucleotide labeling conditions and at the end of the oligonucleotide bound to core proteins Previously described. NF B ? bands were CONFIRMS by competition with a 100-fold excess of unlabeled probe or best. 2.7. I in vitro kinase assay ? B To assay the Kinaseaktivit t the kinase I ? B subunits, the whole cell lysates of cells and RAW264.7, DMSO with LPS stimulation and coxibs embroidered LPS in 50 mM Tris-HCl, 100 M NaCl, 10 mM MgCl2, 1 mM DTT, 10 mM NaF, 1 mM Na3VO4, 0.25 M Cantharidins acid produced. 100 g of cell lysate fra Tasks was charged with 10 M ATP, 1 Ci ATP and incubated 1 g GST IB ? substrate for 30 min at 30 Glutathionesepharose beads were added to the reaction mixture incubated at room temperature for 1 hour to terminate one end of the shaking, and then washed three times with PBS. The beads were treated with a L Centrifuged solution of 2 SDS, at 14,000 g for 10 min and the radioactivity t In the supernatant was determined in a Beckman LS6000LL cooked. Unstimulated cells were used to calculate the fold increase in LPS-treated cells in the presence or absence of coxibs. 2.8 Identification of metabolites selenocoxibs Two milligrams of celecoxib, selenoc