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Functional studies of dephosphorylated and deglycosylated adenoviral vector-derived IGFBP-5.a, untreated IGFBP-5 (lane 1), Antarctic phosphatase-treated IGFBP-5 (lane 2), and neuraminidase- and O-glycosidase-treated IGFBP-5 (lane 3) were compared in Western immunoblotting (WIB) and 125I-IGF-I ligand blotting followed by autoradiography (WLB). b–f, functional comparisons of untreated (▪), dephosphorylated (♦), and deglycosylated (•) IGFBP-5. b, heparin-bound IGFBP-5 was eluted with a linear NaCl gradient from 0 to 1 m. Functional studies of dephosphorylated and deglycosylated adenoviral vector-derived IGFBP-5.a, untreated IGFBP-5 (lane 1), Antarctic phosphatase-treated IGFBP-5 (lane 2), and neuraminidase- and O-glycosidase-treated IGFBP-5 (lane 3) were compared in Western immunoblotting (WIB) and 125I-IGF-I ligand blotting followed by autoradiography (WLB). b–f, functional comparisons of untreated (▪), dephosphorylated (♦), and deglycosylated (•) IGFBP-5. b, heparin-bound IGFBP-5 was eluted with a linear NaCl gradient from 0 to 1 m. Elution fractions were collected (x axis) and assayed for IGFBP-5 by RIA (y axis). c–f, solution binding analysis of increasing concentrations of IGFBP-5 (0.025–5 ng) to 125I-IGF-I tracer (c), 125I-IGF-II tracer (d), and 125I-ALS in the presence of either IGF-I (e) or IGF-II (f). Binding is expressed as a percentage of total tracer bound corrected for nonspecific binding. B/T, bound/total radioactivity. Mark E. Graham et al. Mol Cell Proteomics 2007;6: © 2007 The American Society for Biochemistry and Molecular Biology
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