Figure 4

2D Western blot analysis of species of osteoarthritis (OA) synovial fluid fibronectin (FN) that contain sequences from the N-terminal heparin-binding domain (HBD). Samples of OA synovial fluid (5 μl) were subjected to isoelectric focusing in linear pH gradients followed by reduced 5% SDS–PAGE and Western blot transfer analysis, using anti-N-terminal-HBD mAb 1936 followed by iodinated secondary antibodies. Sample numbers are shown in the right upper corner of each panel. Except for sample nine, blots resulting from pH 4–7 first-dimension isoelectric focusing are presented. The pH 3–10 gradient used for sample nine (i) permitted detection of an ~130-kDa species which was also evident in the three other samples (OA samples 3, 5, and 8) that were submitted to pH 3–10 gradients (not shown). A portion of each synovial fluid sample (5 μl) was submitted to 1D electrophoresis in a lane at the left of each SDS–PAGE gel, and asterisks denote the approximate positions of migration of the ~200+- and ~170-kDa species in these lanes. At least part of the staining of material that migrated as a diffuse band at or near the dye-front in 1D lanes appeared to be nonspecific, since similar staining was present in 1D Western blot analysis of unconcentrated synovial fluid samples in the absence of primary mAbs (not shown). (a) Schematic diagram of the typical 2D migration of three predominant species of synovial fluid FN bearing sequences from the N-terminal HBD: (1) ~170-kDa (major cluster denoted by brackets facing upward): Eight of the nine OA samples contained between two and six ~170-kDa subspecies that migrated as a nearly horizontal array of spots in the cathodic half of the first dimension (pI ~6.0 to ~7.0). In sample number 2 (c), little or no such staining of a ~170-kDa species could be detected, and this correlated with an absence of staining of this species in the 1D lane. Additional ~170-kDa material that migrated much closer to the anode (pI ~4.3) was detected in samples 4 (arrowhead pointing to the right) and 9 (not visible in the pH 3–10 blot in panel i). A species possessing an apparent molecular weight slightly greater than 170-kDa (~180-kDa) was detected as a small spot beneath the cathodic aspect of the ~200+ kDa cluster in samples 1, 3, 4, 7, and 8 (diagonal arrows pointing upward and to the left). (2) ~185-kDa (denoted by small brackets facing downward): OA samples 1 and 3 (b,d), and 5 and 9 (f,g) (blots/exposures not shown) contained an additional fragment species, comprising between one and four faint spots. Similar to the ~170-kDa species, these forms migrated as a near-horizontal array of spots, but farther toward the anode and more slowly (Table 1). (3) ~200+ kDa (denoted by large brackets facing downward): This was detected in all OA samples tested, typically as a large and poorly defined cluster that migrated in the right upper quadrant of each blot. Additional material of ~200+ kDa that migrated farther toward the anode than the major 'cluster' is evident in samples 2, 4, 5 and 9 (c,e,f,i) (short arrows pointing toward the right) (see Table 1). Autoradiogram exposure times were 5 days for samples 1, 3, 5, and 8; 6 days for samples 2, 4, and 7; and 10 days for sample 9. A blot of OA sample 6 is not included in this figure, but can be seen in Figure 6.