Figure 2

Determination of gelsolin isoform in synovial fluid (SF) by immunoblotting. (a) Schematic of the six-domain plasma gelsolin (pGSN) molecule. The respective epitopes for antibodies used are indicated. The 2c4 antibody recognizes the C-terminal half of the GSN molecule and thus both cytoplasmic gelsolin (cGSN) and pGSN. The α-pGSN antibody recognizes the N-terminal plasma extension specific for pGSN. Matrix metalloproteinase-3 (MMP-3) cleavage sites [33] are also indicated. (b) Identical samples were run on two separate gels and probed with rabbit IgG recognizing only the plasma isoform or the 2c4 antibody that recognizes both cytoplasmic and plasma isoforms. Band density was measured by Scion Image 1.62a software, and densities obtained for the two antibodies were compared. Data from representative immunoblots are shown. The relative band intensity is higher for the α-pGSN antibody for all of the SF samples and the plasma sample but much lower for the platelet lysate, which contains mainly the cGSN isoform. This indicates the specificity of the α-pGSN antibody for the pGSN isoform and shows that the main isoform in SF is pGSN (n = 3; 24 SF samples were analyzed). RA, rheumatoid arthritis; rhpGSN, recombinant human plasma gelsolin.