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Supplemental Material METHODS <strong>Schulze</strong> et al. NO regulates thioredoxin through Txnip-Supplemental Material – R2; page 1 Cell Culture. Primary cultures of RPaSMC were prepared from adult Sprague-Dawley rats as previously described, <strong>and</strong> passages between 4 <strong>and</strong> 9 were used for experimentation. RPaSMC were maintained in the RPMI 1640 medium supplemented with 10% NuSerum (Collaborative Biomedical Products, Bedford, MA), penicillin, <strong>and</strong> streptomycin. RPaSMC were exposed to the NO-donor compounds S-nitroso-glutathione (GSNO, 100 µM), PAPA NONOate (NOC-15, 500 µM) <strong>and</strong> S-nitroso-N-acetylpenicillamine (SNAP, 100 µM); 1H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (ODQ, 10 µM), an inhibitor of guanylate cyclase, 1 for different time intervals. 293 cells were plated in DMEM containing 10% FCS, penicillin <strong>and</strong> streptomycin. Transfection of cells was performed at 70% confluence followed by further incubation for 48 h to allow stable protein expression. Northern Analysis. RNA was extracted from RPaSMC using the Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA). 15 µg of cellular RNA was fractionated in 1.5% agarose- formaldehyde gels containing ethidium bromide. RNA was transferred to MAGNA CHARGE membranes (Micron Separations INC., Westboro, MA) <strong>and</strong> crosslinked by ultraviolet light. Membranes were hybridized overnight at 42ºC with 32 P-radiolabeled specific cDNA probes. cDNAs were synthesized using the following oligonucleotides: thioredoxin, 5’– AGC AGC CAA GAT GGT GAA GCA GA -3’ <strong>and</strong> 5’ – CTC CAG AAA ATT CAC CCA CC -3’; Txnip, 5’ –TCT GCC AAA AAG GAG AAG AAA G - 3’ <strong>and</strong> 5’ –GGC GTA CAT AAA GAT AGG- 3’; <strong>and</strong> thioredoxin reductase, 5’– GGC CTC GAC GTC ACT GTA AT -3’ <strong>and</strong> 5’ – TTC CAA TGG CCA GAA GAA AC -3’. cDNA identity was confirmed by sequence analysis. Membranes were washed in a solution containing 3 mM sodium citrate, 30 mM sodium chloride, <strong>and</strong> 0.1% sodium dodecyl sulfate (SDS) Downloaded from http://atvb.ahajournals.org/ by guest on July 30, 2013
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