Journal: PLoS ONE

Article Title: Cyr61/CCN1 Displays High-Affinity Binding to the Somatomedin B 1–44 Domain of Vitronectin

doi: 10.1371/journal.pone.0009356

Figure Lengend Snippet: ( A ) Structure of CCN family members. CCN1 (Cyr61, represented in the figure), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) have a shared structure consisting of a secretory signal peptide (SP), an IGFBP domain (Module I), a von Willebrand type C domain (vWC, Module II), a thrombospondin-1 domain (TSP, Module III) and a cysteine knot (CT, Module IV) domain. Domains are linked by hinge regions susceptible to protease cleavage. Modified from reference . ( B ) Clustal alignment of CCN family members with IGFBP-3 and -5 indicates several conserved residues (*). ( C ) and ( D ) SDS-PAGE for IGFBP-1 to -5, and Cyr61, respectively. Two µg of proteins were loaded in a 4–12% NU-PAGE gel, which was Coomassie blue stained. ( E ) SPR experiments: proteins (100 nM) were used as analytes for immobilized VTNC. Binding levels at stability was used as a value for comparison. RU, resonance units. ( F ) Specificity of Cyr61 (30 nM) tested by solid-phase binding assays: proteins were immobilized as the following densities/well: VTNC (25 ng), fibrilar collagen (50 ng), ixolaris (75 ng), soluble collagen and laminin (100 ng), followed by incubation with Cyr61 (30 nM). Bound-Cyr61 was identified using anti-Cyr61 monoclonal antibody. ( G ) Cyr61 does not inhibit enzyme activity. Cyr61 (300 nM) was incubated with different enzymes and fluorogenic substrate hydrolysis was followed as indicated in . SEM are not depicted but were less than 5% (n = 3).

Article Snippet: Human placenta soluble collagen Type I was from BD Biociences (Bedford, MA, USA), Horm type I fibrilar collagen was from Chrono-log (Havertown, PA, USA) and laminin was from Upstate Biotechnologies (Charlottesville, VA, USA).

Techniques: Modification, SDS Page, Staining, Binding Assay, Incubation, Activity Assay