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3543094188
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note
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ext antibodies to extracellular epitopes of the leptin receptor.
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3543116391
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note
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int, Ulex europaeus agglutinin I, or normal rabbit serum. After incubation with primary antibodies, tissue sections were developed with secondary horseradish peroxidase-conjugated goat antibody to rabbit IgG with a Vectastain Elite ABC kit (Vector Labs). The slides were counterstained with hematoxylin.
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3543142129
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int (9) or with RC20 recombinant anti-phosphotyrosine (Transduction Laboratories, Lexington, KY).
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24
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705) rabbit polyclonal antibody, was used for detecting the presence of Stat3 and phospho-Stat3 in the cell lysates.
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25
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3543066072
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2, 20 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM phenytmethylsulfonyl fluoride, and a mixture of protease inhibitors]. After a 15-min incubation on ice, cells were homogenized, and nuclei were collected by centrifugation. The pellet was resuspended in low-salt buffer adjusted to 0.5 M KCl, extracted for 30 min at 4°C, and centrifuged at 25,000g for 30 min. Supernates were collected, dialyzed against buffer made 0.1 M with KCl, and divided into small portions kept at -80°C until used.
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32P-labeled probe for 20 min at room temperature in binding buffer [20 mM tris-HCl (pH 7.9), 50 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, bovine serum albumin (50 μg/ ml), and polydeoxyinosine/polydeoxycytosine (2 μg/ ml)]. Electrophoresis sample buffer was added to each sample before separation in a nondenaturing gel (89 mM tris, 89 mM boric acid, 10 mM EDTA, and 4.95% acrylamide). Gels were dried and exposed to autoradiography.
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note
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2 incubator at 37°C for 2 to 5 min, allowing them to solidify. Growth medium supplemented with endothelial cell growth supplement with or without human recombinant leptin was then added to each dish. Leptin was replenished every 24 hours. HUVECs were allowed to form tubelike structures for 2 days in culture and were then frozen and sectioned to a thickness of 40 μm. For immunofluorescence analysis, the sections were fixed with acetone (-20°C) and stained with tetramethylrhodamine isothiocyanate-labeled Ulex europaeus agglutinin I (0.5 μg/ml) and 4′,6′-diamidino-2-phenylindole (DAPI) (0.0001%).
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3543057888
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note
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Abbreviations for the amino acid residues are as follows: C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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39
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3543055514
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note
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We thank T. Buckholz and J. Kupcho for the synthetic peptides and conjugates, L. Friedman for the anti-leptin receptor, L. Benson and G. Davis for HUVEC cultures, D. O'Connor for help in migration studies, and J. Pober for laboratory resources.
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