Figure 1. Coimmunoprecipitation of IGFBP-5 and PAI-1 Figure 1. Coimmunoprecipitation of IGFBP-5 and PAI-1. A, IGFBP-5, 50 ng/ml, was incubated with PAI-1, 100 ng/ml, and anti-PAI-1 antibody, 1:5000 dilution, for 16 h at 4 C. At that time, immunoprecipitation was conducted by adding protein A sepharose, as described in Materials and Methods. The pellets were dissolved in Laemmli sample buffer and the products electrophoresed through a 12.5% gel, then transferred to Immobilon membranes and immunoblotted using a 1:1000 dilution of anti-IGFBP-5 antiserum. The results show that, in the presence of PAI-1, IGFBP-5 could be coimmunoprecipitated, whereas the anti-PAI-1 antibody did not immunoprecipitate IGFBP-5 without PAI-1 being added. PAI-1 did not react with the IGFBP-5 antibody. B, ¹²⁵I-IGFBP-5, 50,000 cpm/ml, was incubated with anti-PAI-1 antiserum (1:5000) and PAI-1 (100 ng/ml). In the presence of PAI-1, an abundant ¹²⁵I-IGFBP-5 band was detected. In the absence of the PAI-1 antibody or unlabeled PAI-1, minimal ¹²⁵I-IGFBP-5 was immunoprecipitated. The asterisk denotes an incubation mixture that contained excess unlabeled IGFBP-5 (100 ng/ml). Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society

Figure 3. The effects of IGFBP-5 peptides on PAI-1 and IGFBP-5 binding Figure 3. The effects of IGFBP-5 peptides on PAI-1 and IGFBP-5 binding. Radiolabeled IGFBP-5, 50,000 cpm/ml, was incubated with unlabelled PAI-1, 100 ng/ml, and 100 μg/ml of one of four peptides, lanes 2–5, that contained various sequences within the IGFBP-5 molecule (9 ). After a 14-h incubation, the products were coimmunoprecipitated and processed by SDS-PAGE and transferred to Immobilon. The figure is an autoradiogram showing the amount of ¹²⁵I-IGFBP-5 that was coimmunoprecipitated. Lanes 1–5, ¹²⁵I-IGFBP-5; lane 2, peptide A; lane 3, peptide B; lane 4, peptide C; lane 5, peptide D. The radiolabeled band with an M_r estimated of 18 kDa shown in lanes 1, 4, and 5 is a fragment of radiolabeled IGFBP-5. Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society

Figure 2. The effects of glycosaminoglycans on IGFBP-5/PAI-1 association. ¹²⁵I-IGFBP-5 was incubated without, lane 1, or with 100 ng/ml of PAI-1 (lanes 2–7), then immunoprecipitated as described in Materials and Methods. After immunoprecipitation, the pellets were dissolved in Laemmli sample buffer and the products electrophoresed, then transferred to Immobilon membranes and autoradiography performed directly; lanes 2–7, ¹²⁵I-IGFBP-5; lanes 3–7, ¹²⁵I-IGFBP-5 plus various glycosaminoglycans (0.1 μg/ml). Lane 3, heparin; lane 4, heparan sulfate; lane 5, chondroitin sulfate A; lane 6, chondroitin sulfate B; lane 7, chondroitin sulfate C. The radiolabeled band shown in lane 1 that migrates with an M_r estimate of 16 kDa is a fragment of radiolabeled IGFBP-5. Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society

Figure 4. Coimmunoprecipitation of IGFBP-5 mutants and PAI-1 Figure 4. Coimmunoprecipitation of IGFBP-5 mutants and PAI-1. One hundred nanograms per milliliter of each IGFBP-5 mutant was incubated with 100 ng/ml of PAI-1 and the products immunoprecipitated using anti-PAI-1 antiserum as described previously. After SDS-PAGE, the products were transferred to an Immobilon membrane, then ligand blotted using ¹²⁵I-IGF-I as described in methods. Lane 1, wild-type IGFBP-5, no PAI-1; lanes 2–8, PAI-1 plus IGFBP-5 or mutants. Lane 2, wild-type IGFBP-5; lane 3, K134A/R136A; lane 4, R202A/K206A/R207A; lane 5, R201A/K202N/K206N/K208N; lane 6, K211N; lane 7, R201N/K202N; lane 8, K211N/R214A/K217A/R218A. The results of Phor Image analysis expressed as scanning units were: lane 1, 2,237; lane 2, 190,511; lane 3, 204,125; lane 4, 170,867; lane 5, 76,748; lane 6, 250,813; lane 7, 223,452; lane 8, 146,656. Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society

Figure 5. The effect of PAI-1 on IGFBP-5 proteolysis Figure 5. The effect of PAI-1 on IGFBP-5 proteolysis. IGFBP-5, 50 ng/ml, was incubated with IGFBP-5 protease that had been purified from human fibroblast conditioned medium. After a 14-h incubation, the products were electrophoresed and analyzed by immunoblotting. Lanes 1–4, IGFBP-5 plus protease; lane 2, vitronectin, 10 μg/ml; lane 3, PAI-1, 10 μg/ml; lane 4, PAI-1 plus vitronectin. The 22-kDa band, which is the major cleavage product of this protease, is shown by an arrow. Scanning unit values for the intact IGFBP-5 bands were; lane 1, 87,644; lane 2, 107,781; lane 3, 144,811; and lane 4, 123,322. Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society

Figure 6. The effect of vitronectin on the binding of PAI-1 to IGFBP-5 Figure 6. The effect of vitronectin on the binding of PAI-1 to IGFBP-5. A, ¹²⁵I-IGFBP-5 and PAI-1 (100 ng/ml) were incubated with anti-PAI-1 antisera and increasing concentrations of vitronectin. After a 6-h incubation, the products were immunoprecipitated, electrophoresed, and the gel analyzed by autoradiography. Lane 1, ¹²⁵I-IGFBP-5 alone; lanes 2–5, ¹²⁵I-IGFBP-5 plus PAI-1; lane 3, vitronectin (1 μg/ml); lane 4, vitronectin (0.1 μg/ml); lane 5, vitronectin (0.01 μg/ml). Phosphor Image intensity units were: lane 1, 46,673; lane 2, 98160; lane 3, 65,869; lane 4, 75077; and lane 5, 81,264. B, ¹²⁵I-IGFBP-5 and vitronectin (100 ng/ml) were incubated with antivitronectin antiserum (1:5000), then the products immunoprecipitated as in panel A. Lane 1, ¹²⁵I-IGFBP-5 alone; lane 2, vitronectin (100 ng/ml); lane 3, PAI-1 (100 ng/ml); lane 4, PAI-1 (1000 ng/ml). Phosphor Image intensity units were lane 1, 31,342; lane 2, 86,192; lane 3, 44,360; and lane 4, 36,444. Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society

Figure 7. Scatchard plot. ¹²⁵I-IGFBP-5 (30,000 cpm/tube) was incubated with increasing concentrations of unlabeled IGFBP-5 (5–1000 ng/ml) and PAI-1, 100 ng/ml. The products were immunoprecipitated as described in Materials and Methods and bound ¹²⁵I-IGFBP-5 determined directly by gamma counting. Nonspecific binding was defined as the cpm bound in the presence of 10 μg/ml of unlabeled IGFBP-5 and was less than 4% of the total binding. Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I* Endocrinology. 1997;138(7):2972-2978. doi:10.1210/endo.138.7.5230 Endocrinology | Copyright © 1997 by The Endocrine Society