Localization of WARP and collagen VI using electron microscopy.

<p>EM analysis on native supramolecular fragments isolated from human articular cartilage. Sheep antisera against WARP (18-nm gold particles; black arrow heads) (panels a–c) and a rabbit polyclonal anti-collagen VI (panels a and b) or monoclonal antibody against collagen VI (panel c) (12-nm gold particles; white arrowheads) was used. A secondary antibodies only control is shown in panel d. Scale bars: 100 nm.</p

Solid phase analysis of WARP/collagen VI interaction.

<p>a, WARP binding to collagen VI tetramers. Coated collagen VI was incubated with recombinant WARP. Data are means of triplicate determinations +/− SE. <i>Inset</i>, Scatchard analysis of binding data reveals an apparent kDa of 22 nM. b, Reciprocal solid phase binding experiment showing collagen VI binding to coated WARP dimer but not WARP multimers. Representative curves are shown. c, WARP binds to both pepsinized collagen VI tetramers (black diamonds) and pepsinized intact collagen VI (white squares).</p

Collagen VI suprastructure analysis.

<p>A, Collagen VI suprastructures were isolated from human articular cartilage extracts using superparamagnetic immunobeads coupled to collagen VI antibodies. The isolated suprastructures were then doubly immuno-labeled with sheep anti-WARP antiserum (18-nm gold particles; black arrowheads) and rabbit anti-collagen VI antibody (small gold particles; white arrowheads). WARP is present in collagen VI suprastructures. Scale bar is 100 nm.</p

WARP binds to collagen VI <i>in vitro</i>.

<p>Isolated collagen VI microfibrils (shown in a) were mixed with recombinant WARP. Globular structures representing WARP are marked with arrowheads and visualized by negative staining (shown in b). Biotinylated recombinant WARP was visualized by gold labeled streptavidin as shown in panel c (5-nm gold particles). WARP is present on the collagen VI microfibrils (panel d) visible as structures close to the globular domains of the collagen VI microfibrils (bound WARP; black arrowheads). These structures are absent in the control experiments where WARP is omitted (see a). Biotinylated recombinant WARP (5-nm gold particles) bound near the junction between helical and globular domains of collagen VI microfibrils (panels e–g). Scale bars: 100 nm in a, b and d, 50 nm in e, 25 nm in c and f, and 10 nm in g.</p

Representative ultrathin sections of articular cartilage of adult mice co-stained for WARP (18-nm gold particles; black arrow heads) and collagen VI (12-nm gold particles; white arrowheads).

<p>Scale bars: 100 nm.</p

Analysis of collagen VI, WARP and perlecan in human articular cartilage.

<p>Immuno-gold EM was conducted on native supramolecular fragments isolated from human articular cartilage for WARP (18-nm gold particles; black arrowheads), perlecan (12-nm gold particles; white arrowheads) and collagen VI (6-nm gold particles, arrows). All three components are part of complexes at the suprastructural level (shown in a). The magnified image (shown in b) shows these complexes in close contact to banded collagen fibrils. Scale bars: 100 nm (panel a) and 200 nm (panel b).</p

Immunohistochemical localization of WARP and collagen VI in articular cartilage.

<p>A, Superficial zone human articular cartilage was stained for collagen VI (panels a, d and g) and WARP (panels b, e and h). The merged images show clear co-localization of collagen VI and WARP in the pericellular environment (panels c, f and i). The scale bar shown in panel (a) is 100 µm. B, Mouse tibial articular cartilage stained with collagen VI (panels a and d) and WARP antisera (panels b and e). Merged images show co-localization of collagen VI and WARP in the chondrocyte pericellular matrix (panels c and f). Scale bar in panel (a) is 200 µm and in (d) 50 µm.</p