Figure 3

Nonreduced analysis of species of osteoarthritis (OA) synovial fluid fibronectin (FN) that bear sequences from the N-terminal heparin-binding domain. (a) OA sample 6 was subjected to gelatin affinity isolation, and the starting material ('SM') and flow-through ('FT') fractions were submitted to 5% nonreduced SDS–PAGE followed by Western blot analysis in duplicate using monoclonal antibodies (mAbs) specific for the N-terminal heparin-binding domain ('anti-N-term') or the gelatin-binding domain (GBD) (mAb 1892, 'anti-GBD'). In the starting material and the flow-through fraction, the anti-N-terminal mAb recognized a fragment species with mobility expected of a reduced protein of ~140 kDa ('F'), in addition to dimeric ('D') and monomeric ('M') species. Although staining of all three species was less in the flow-through fraction than in the starting material, the reduction in staining of the dimeric and monomeric forms was substantially greater than for the fragment species. In contrast, the anti-GBD mAb produced staining of species with mobility expected of dimeric ('D') and monomeric ('M') FNs but did not stain a fragment species in the starting material or the flow-through fraction. The two pairs of lanes were derived from one autoradiogram, which was exposed overnight. Similar results, in which dimeric and monomeric species of FN were stained by anti-GBD mAb to the exclusion of the smaller fragment species, were obtained for OA samples 1, 4, and 9 (not shown). (b) Purified 30-kDa N-terminal heparin-binding ('30 K') and 45-kDa gelatin-binding ('45 K') fragments of human FN (2.5 μg each), as well as the 170-kDa-enriched fraction derived from OA synovial fluid sample 6 ('170 K') (5 μl) [15], were subjected to duplicate 4–15% nonreduced SDS–PAGE and Western blot analysis using mAbs to the N-terminal heparin-binding domain (left) or to the GBD (right). The anti-N-terminal mAb produced staining of the 30-kDa fragment and a species with migration expected of a reduced protein of ~140-kDa within the 170-kDa-enriched fraction, but failed to stain the 45-kDa fragment. In contrast, the anti-GBD mAb produced bright staining of the 45-kDa fragment, but failed to stain the 30-kDa fragment or material in the 170-kDa-enriched fraction. The 30-kDa fragment migrated faster than expected from the positions of migration of reduced molecular weight standards shown to the left of each panel, possibly reflecting the effect of maintenance of type I repeat intrachain disulfide bonds upon conformation under nonreducing conditions. Autoradiagram exposure times were 4 hours for the 30 K and 45 K lanes, and overnight for the 170 K lanes.