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Fig. 6. Alanine scanning of HN peptide showing important amino acids for IGFBP-3 binding (Upper). pAla-scanned HN-FLAG plasmids, pHN, or pL9R-HN were transfected in triplicate to F11 cells. F6A and K21A-mutant HN peptides did not pull down IGFBP-3. pHN and vector alone were used as positive and negative controls, respectively. As positive controls, synthetic HN peptide was used instead of cell lysate (HN), and rhIGFBP-3 alone was applied to the gel (BP3). Competitive cross-linking analysis showing displacement of ¹²⁵I-HN from IGFBP-3 with excess cold HN and F6A-HN (Lower). ¹²⁵I-HN was cross-linked to IGFBP-3 (1 mg) alone or in the presence of competing cold HN or F6A-HN at 0.1, 1, and 2 m g. ¹²⁵I-HN bound to IGFBP-3 was found at »36 kDa (lane 3). One and 2 mg of cold HN completely displaced ¹²⁵I-HN from IGFBP-3 (lanes 4 and 5), and 0.1 mg of HN reduced IGFBP-3 bound ¹²⁵I-HN by » 45% in pixel volume (lane 6). Whereas 2 mg of F6A-HN showed moderate displacement (lane 7), 1 and 0.1 m g of F6A-HN did not (lanes 8 and 9). Lanes 1 and 2 show ¹²⁵I-HN alone with DSS cross-linker. Results are a representative of three independent experiments.