Figure 3

Immunoblots of plasma gelsolin (pGSN) cleavage products generated by matrix metalloproteinase (MMP).(a) The catalytic domain of MMP-3 (cMMP-3) was incubated with recombinant human pGSN (rhpGSN) at a 2:1 cMMP-3/pGSN ratio for various time points at 37°C. pGSN is cleaved and cleavage products of approximately 42 to 46 kDa are detected after 30 minutes of incubation using the 2c4 antibody, which recognizes the C-terminal half of GSN (Figure 2a) (n = 3). (b) cMMP-3 was incubated with human plasma from healthy controls for various time points at 37°C. More GSN cleavage products are visible with increasing incubation time, indicating cleavage and showing that cleavage products generated by MMP-3 should be detected in plasma if present at high enough quantities. (Note that hrpGSN or plasma incubated without cMMP-3 for 180 minutes does not show breakdown products.) The amount of pGSN added in (a) and (b) corresponds approximately to the average amount of pGSN added in (c). (c) Immunoblots of plasma and synovial fluid (SF) from randomly selected rheumatoid arthritis (RA) patient samples. MMP cleavage products are approximately 42 to 46 kDa in size and are recognized by 2c4 IgG (a, b) [33]. No cleavage products were observed in the patient samples (36 different plasma and SF samples were tested in three independent experiments). Representative immunoblots are shown.