Bloch and Richard T. Lee P. Christian Schulze, Heling Liu, Elizabeth ...

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Schulze et al. NO regulates thioredoxin through Txnip-Supplemental Material - R1; page 2 at 65 ºC and exposed to X-ray film. By staining 28S and 18S ribosomal RNA with ethidium bromide, equal loading of RNA on gels was confirmed. Quantitative real-time PCR. Txnip gene expression was analyzed by real time PCR (LightCycler, Roche Applied Science) using specific oligonucleotides: rat Txnip, 5'- CAAGTTCGGCTTTGAGCTTC-3' (sense) and 5'-GCCATTGGCAAGGTAAGTGT-3' (antisense); rat β-tubulin, 5'-CATCCAGGAGCTCTTCAAGC-3' (sense) and 5'- CGCCTTAGGCCTCTTCTTCT-3' (antisense). Western Analysis. RPaSMC were washed twice with 10 ml of ice-cold phosphate-buffered saline (PBS) and harvested by scraping with a rubber policeman into buffer that contained 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenyl-methlsulfonyl fluoride. Cell membranes were disrupted by passing through a 22-gauge needle 10 times. Cell extracts were centrifuged at 10,000 x g for 30 min at 4 ºC. Cell supernatants containing 50 µg of protein were subjected to 8% sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membranes (Micron Separations Inc., Westboro, MA). The membranes were blocked in phosphate-buffered saline containing 5% non-fat milk at room temperature for 1h and then incubated with antibodies directed against Txnip, thioredoxin reductase or thioredoxin. After incubation with horseradish peroxidase-conjugated secondary antibodies, positive immunoreactivity was visualized using enhanced chemiluminescence. Nuclear run-off experiments. Nuclear run-off assays were performed as previously described. 2 cDNA probes were created using specific oligonucleotides: rat Txnip, 5'-CAA GTT CGG CTT TGA GCT TC-3' (sense) and 5'-GCC ATT GGC AAG GTA AGT GT-3' (antisense); rat β-tubulin, 5'-CAT CCA GGA GCT CTT CAA GC-3' (sense) and 5'-CGC CTT AGG CCT CTT CTT CT-3' (antisense); rat thioredoxin sense 5´-GCT GAT CGA GAG CAA GGA AG-3 and antisense 5´-TCA AGG AAC ACC ACA TTG GA-3´. Equal amounts of cDNA (5ug) were blotted Downloaded from http://atvb.ahajournals.org/ by guest on July 30, 2013
Schulze et al. NO regulates thioredoxin through Txnip-Supplemental Material - R1; page 3 onto nitrocellulose membranes. Identical numbers of control and GSNO-treated cells were used for the isolation of nuclei and preparation of [ 32 P]UTP-radiolabeled transcripts. Signals were visualized by autoradiography. Specific radioactive signal intensity (as counts per minute) was determined for each probe individually using a scintillation counter and normalized over signal intensity of the housekeeping gene β-tubulin. mRNA stability. Cells were pretreated with actinomycin D (5 µg/mL for 30 min) before incubation with and without GSNO stimulation (100 µM) for varying durations. RNA was extracted, and Txnip gene expression was measured by quantitative PCR and normalized for expression of β- tubulin as described before. 3 Plasmid construction and transient transfection experiments. The human Txnip promoter region including -1777 bp upstream of the ATG start codon was cloned from human genomic DNA using primers 1-6 (Table 1). To generate the Txnip promoter constructs, PCR products were extracted and cloned into a firefly luciferase reporter vector pGL3-Basic Vector (Promega). The transcriptional activity of the promoter constructs was assessed under stimulation with GSNO at 5.6 mM and 22.4 mM glucose. Luciferase activity was determined using the Dual Light assay kit (Tropix). All experiments were repeated five times. Further, full-length human Txnip was cloned into a mammalian expression vector (pcDNA3.1, Invitrogen). Expression plasmids for human wildtype thioredoxin or mutant thioredoxin with a serine replacing cysteine 69 (C69S) were kindly provided by Dr. Judith Haendeler (Molecular Cardiology, University of Frankfurt, Germany). These vectors express a thioredoxin-Xpress-tag fusion protein. Complete sequence identity was confirmed by sequencing analysis. Cells were transfected using FUGENE transfection reagent (Roche Applied Biosystems) and were studied 48 hours later. Equal amounts of empty expression plasmids served as control vectors. Downloaded from http://atvb.ahajournals.org/ by guest on July 30, 2013
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