Figure 5

2D Western blot analysis of species of rheumatoid arthritis (RA) synovial fluid fibronectin (FN) that contain sequences from the N-terminal heparin-binding domain. RA synovial fluid samples 10–19 were subjected to linear pH 4–7 first-dimension isoelectric focusing followed by reduced second-dimension 5% SDS–PAGE. After transfer, membranes were stained with mAb to the N-terminal heparin-binding domain followed by iodinated secondary antibodies. Sample numbers are shown in the right upper corner of each panel. Species of ~200+ kDa (large brackets facing down) that migrated at a position similar to corresponding species in OA samples (see Fig. 4) were evident in all 10 RA samples. An additional cluster of material of ~200+ kDa, denoted by short arrows pointing toward the right, was evident in samples 11–13 and 15–19 (see Table 1). This material was streaked upward in samples 12, 16, 17, and 18. Definitive staining of ~170-kDa species (large brackets facing up) was evident in samples 10–15, 17, and 18. Additional ~170-kDa material that migrated much closer to the anode (pI ~4.3) than the major cluster was evident in RA sample 17 (h) (arrowhead pointing toward the right). An additional species that possessed a molecular weight of approximately 180 kDa was detected as a spot beneath the cathodic aspect of the cluster of ~200+ kDa in samples 11–15 (diagonal arrows pointing upward and to the left) (see Table 1). An ~185-kDa species (small bracket facing down) is evident in samples 10, 11, 13–15, and 18. Autoradiograms were exposed overnight for sample 19, 2 days for sample 18, 4 days for samples 10, 13, and 16, 5 days for samples 11, 14, 15, and 17, and 6 days for sample 12. No definitive staining of ~170- or ~185-kDa species was observed in samples 16 or 19, even after exposure times as long as 10 days.