Phosphate dehydrogenase and uPAR. The NART sequences were specific primers GAPDH sense, 5 TTG TTG CCA TCA ATG ACC CC 3, GAPDH antisense, 5 TGA CAA GGT CGT AGT TGA GG 3, meaning uPAR, 5 CAC CAG CGT GCG GTG CTT GG 3, uPAR and antisense 5 TGT TCT TCA GGG CTG CGG CA 3 The PCR conditions were denaturation at 94 for 30 s, annealing at 58 for 30 s and Verl EXTENSIONS at 72 for 45 s The products were electrophoresed in 1.5% agarose gel containing ethidium bromide subjected. Northern blot analysis. Total RNA extraction and Northern blot hybridization were performed as previously described. Each cDNA probe was deoxyribonucleoside triphosphate using the technique with the Rediprime labeling system randompriming radioactively labeled. The nylon membranes were interviewed to R Ntgenfilm exposed. Western blot analysis. Cells pretreated with 0,500 g / ml were of nicotine for various times in phosphate-buffered saline Solution, gel St with trypsin-EDTA and stored at 0 to ben CONFIRMS. The protein was extracted with RIPA buffer and protease inhibitors, benzamidine, trypsin inhibitor, sodium orthovanadate. Fifty micrograms of protein was then separated by SDS-10% polyacrylamide gel electrophoresis and on polyvinylidene fluoride membranes. The membranes were incubated in a PBS-L Measurement solution with 5% nonfat drymilk, incubatedwith prime Ren Antique Body in Blockierungsl Blocked overnight at 4 and three times with 0.1% Tween 20 in Tris-buffered saline Solution at intervals min ligand of 10. Peroxidaseconjugated secondary Ren Antique Body was used to detect horseradish immunoreactive proteins by chemiluminescence. The following antique body were used: anti-uPAR, anti IB, anti-phospho p44/42 MAPK JNK phospho p38MAPK antibody and anti-phospho. The total protein content measured level were bywashing the membrane with a Beizl Solution of 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl for 30 minutes at 50 and the membrane gel was Deleted then either actin and anti-Erk 1 / 2, JNK, anti-2 or anti-p38MAPK monoclonal antibody body is. Measurement of uPAR promoter activity t. Transcriptional regulation of uPARwas by transient transfection of a luciferase reporter construct examined uPAR promoter. The uPAR promoter plasmid pGL3 was kindly provided by Dr. Y. Wang made available. ECV304 cells were seeded and until they reach confluence, 60 70%, and pRL TK and pGL3 uPAR were in cells using FuGENE according to claim manufacturer’s protocol co-transfected. pRL TK was transfected as a contr the house. The cells were incubated in the transfection way for 20 h and 0,500 g / ml of nicotine for 5 The effects of signal inhibitors on the activity h t of the uPAR promoter were by pretreatment of cells with inhibitors for 1 h before addition of nicotine determined. The studies were co-transfection in the presence or absence of K The oligodeoxynucleotides AP 1 or MEK 1, C June I performed  B, I  B or B NF  Feeder Rderungsverfahren kinase. The doppelstr Independent phosphorothioate ODN with the sequences against the AP 1 binding site were synthesized and annealed. The expression vector encoding inactive MEK 1 and c in June was a gift fromDr. N.G. Ahn and Dr. M.J. Birrer, respectively. The dominant negative mutants of IB and I B and NIK   were kindly provided by Dr. DW Ballard, and Dr. WC Gree available.