Fig. 3
Specific increase of citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) in sera in patients with rheumatoid arthritis (RA). a Sera were separated by SDS-PAGE from patients with RA, healthy subjects (HS), and peptide glucose-6-phosphate isomerase-induced arthritis (pGIA) mice, then stained with Coomassie brilliant blue (CBB) (left) or subjected to Western blot analysis using anti-modified citrulline (AMC) antibodies (right). Citrullinated proteins were detected as ~ 120 kDa bands in patients with RA and pGIA mice but in none of the HS. CBB staining is shown to demonstrate loading. Lanes 1–5: HS; lanes 6–11: RA; lane 12: pGIA mice at day 14. b The band intensity of 120 kDa citrullinated protein. Sera from patients with RA (n = 60), HS (n = 30), patients with osteoarthritis (OA) (n = 12), patients with systemic lupus erythematosus (SLE) (n = 15), and patients with Sjögren’s syndrome (SS) (n = 27) subjected to Western blot analysis using AMC antibodies. Citrullinated proteins at 120 kDa were specifically increased in patients with RA. Each symbol represents a single sample. Horizontal bars represent the mean values; vertical bars represent the SEM; and broken line represents the cutoff value. **p < 0.01, N.S. Not significant. c Serum samples from patients with RA and HS separated by two-dimensional PAGE, then stained with CBB (left) or subjected to Western blot analysis using AMC antibodies (right). Citrullinated proteins were detected at ~ 120 kDa and pH 5.3 in patients with RA but not in HS. d The MS spectrum of ITIH4429–438 and modified peptides bearing the citrullinated arginine (R438) in patients with RA. Citrullinated residues identified by the unmodified b3 and b6 ions and the modified y1, y6, and y8 ions confirmed a mass increase of 1.0 Da. The experiments shown in (c) and (d) were performed on at least three different samples with similar results. e Serum samples subjected to Western blot analysis using anti-ITIH4 antibodies (Ab) (left) and AMC antibodies (right). The bands were detected at 120 kDa by anti-ITIH4 antibodies as well as AMC antibodies