Kinetics of binding of extracellular matrix proteins in solution to solid-phase sRAGE.

a<p>Not determined due to undetectable specific binding.</p

Organ biodistribution of intraperitoneally-administered sRAGE or MSA in mice.

<p>Organ biodistribution of mouse sRAGE or MSA (A) 1 hour, (B) 2 hours, (C) 4 hours, and (D) 12 hours after intraperitoneal injection of ∼1 µCi of tracer, corresponding to ∼0.6–1.4 µg of radiolabeled protein. Asterisks indicate statistically significant differences.</p

Clearance of intratracheally-administered sRAGE from mouse lung occurs at the alveolar-capillary interface.

<p>Representative light micrographs/autoradiographs recorded at 20× magnification of sections of lung from mice given mouse sRAGE (or MSA, shown for comparison) i.t. and sacrificed at the indicated time points thereafter. Lung from untreated mice serves as a control for background radiation.</p

Mouse and human sRAGE extinction coefficients.

a<p>Mouse membrane RAGE has a published sequence length of 402 (NP_031451.2).</p>b<p>Human membrane RAGE has a published sequence length of 404 (NP_001127.1).</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088259#pone.0088259-Pace1" target="_blank">[50]</a>.</p

Organ biodistribution of intratracheally-administered sRAGE or MSA in mice.

<p>Organ biodistribution of mouse sRAGE or MSA (A) 1 hour, (B) 2 hours, (C) 4 hours, and (D) 12 hours after intratracheal instillation of ∼1 µCi of tracer, corresponding to ∼0.6–1.4 µg of radiolabeled protein. Asterisks indicate statistically significant differences.</p

sRAGE and MSA used in biodistribution and clearance studies are very pure.

<p>(A) SDS-PAGE separation and Coomassie Brilliant Blue staining of ∼2.5 µg of MSA or mouse sRAGE preparations used for radiolabeling. (B) SDS-PAGE separation and gel autoradiography of ¹²⁵I-labeled MSA and mouse sRAGE.</p

Clearance of intratracheally-administered sRAGE or MSA from mouse lung is rapid and non-proteolytic.

<p>(A) Exogenous mouse sRAGE or MSA persistence in mouse lung as a function of time. (B) SDS-PAGE separation and gel autoradiography of lung homogenates from mice administered i.t. mouse sRAGE or MSA and sacrificed at the indicated time points thereafter.</p

RAGE binds with high affinity to collagen I, collagen IV, and laminin, but not to fibronectin.

<p>Soluble RAGE affinity for collagen I, collagen IV, laminin, and fibronectin was assessed by biolayer interferometry. Soluble RAGE was conjugated to an amine-reactive sensor and dipped into solutions containing collagen I (A), collagen IV (B), laminin (E), or fibronectin (F) for 1800 s, at which point dissociation was initiated by washing in PBS. Binding was measured as a deflection in the wavelength of light passing through the sensor; affinities were calculated by fitting the curves with analysis software. Reciprocal binding was studied with collagen I (C) and collagen IV (D) conjugated to the sensor and sRAGE in solution. Laminin and fibronectin could not be conjugated to the sensor.</p

Cytokine RNA quantity in lung slices.

<p>* = Significant difference between strains.</p><p>IL-1α, IL-1β, and TNF-α levels were all significantly higher in LPS treated RAGE KO lung slices compared to wild type after LPS challenge. IL-6 and MCP-1 were both higher in the LPS treated wild type lung slices compared to LPS treated RAGE KO.</p><p>*significant difference between wild type and RAGE KO (p<0.05).</p><p>Data are mean ±SEM, analyzed by 2-way ANOVA with a Bonferroni post hoc test. (<i>n of 3 per group</i>). Abbreviations IL: interleukin, TNF: tumor necrosis factor, MCP: monocyte chemotactic protein.</p

RAGE KO mice get less inflammation than wild type mice after <i>E. coli</i> pneumonia.

<p><i>E. coli</i> challenged RAGE KO mice had significantly less total cells/mL in BALF compared to wild type mice (A). The lower amount of cells was mainly due to less neutrophils (B, C). Protein in BALF (D) and myeloperoxidase activity of whole lung (E) were also significantly less in RAGE KO than in wild type mice after <i>E. coli</i> challenge. * indicate significant increase/decrease compared to vehicle control for each strain. ** indicate significant difference between wild type and RAGE KO mice. Data are mean ±SEM, analyzed by 2-way ANOVA with a Bonferroni post hoc test (<i>n of 6–8 per group</i>).</p