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note
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An electrophoretic mobility-shift assay (EMSA) was done in buffer containing 50 mM NaCl, 1 mM EDTA 10 mM Tris-HCI (pH 7 5), 5% glycerol, 0 5% Nonidet-P40 (Sigma), 1 mM dithiothreitol, and 20 μg of bovine serum albumin (BSA) for 30 min at 22°C.
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35S-undine 5′-triphosphate (UTPJ-labeled riboprobe for 16 hours. After washing, the slides were coated with autoradiographic emulsion and exposed for 3 weeks. The images were photographed and digitized The hybridization signal of the radiolabeled probe appears as white grains All specimens were observed under dark field illumination after nuclear counterstain with hematoxylin. Immunostainmg for factor VIII-related antigen (4) confirmed that injury was limited to endothelium
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2, 5% glycerol, 2 5 mM Hepes (pH 7.9), 1 μg of polydeoxyinosinic-deoxycytidylic acid, and 20 μg of BSA for 30 min at 22°C Polyclonal antipeptide antibodies to Sp1 and Egr-1 (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear extracts 15 min before the addition of the probe.
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d77mEgr-CAT was constructed with the use of an oligonucleotide bearing the Oligo Bm (12) sequence as the 5′ primer for the polymerase chain reaction. Transfections in BAECs were performed with 10 μg of reporter plasmid and the calcium phosphate protocol (10). The cells were incubated with PMA (100 ng/ml), cotransfected with CMV-Egr-1, or injured with a sterile comb (8), and then incubated for 36 hours at 37°C. CAT activity was assessed by the two-phase fluor diffusion technique (13) and was normalized to the amounts of protein in the cell lysate
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32P-Oligo B (72) for 30 min at 22°C and applied to a running nondenaturing 5% polyacrylamide gel at the times indicated Alternatively, a 1000-fold molar excess of the unlabeled cognate was added after the 30-min incubation and applied to the gel (21).
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13344255452
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note
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32P-Oligo B was incubated with a fixed concentration of Sp1 and decreasing amounts of Egr-1 (Fig 3B, right side) (21).
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40
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13344289071
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Protein immunoblots were analyzed with polyclonal antibodies to Egr-1 and Sp1 (1 2500: Santa Cruz Biotechnology) and to PDGF-B (1:200; Genzyme) Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham) with horseradish peroxidase-linked donkey secondary antiserum to rabbit immunoglobulin at 1 10,000 dilution.
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note
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We thank F J. Rauscher III for recombinant Egr-1, V. P. Sukhatme for CMV-Egr-1, M. A Frosch for critical review of the manuscript, and M. A. Gimbrone Jr. for enthusiastic support. Supported in part by grants from NIH (T.C.) and the American Heart Association (V.L.). L M.K. is a C. J. Martin Postdoctoral Research Fellow (National Health and Medical Research Council of Australia) and a recipient of a J William Fulbright Postdoctoral Research Award T.C. is an Established Investigator of the American Heart Association.
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