Data were normalized relative to the protein concentration in each sample

Data were normalized relative to the protein concentration in each sample. LC-MS Sera from pGIA mice, control mice, patients with RA, and HS were separated by 2D-PAGE, and gels were stained with BAY-598 GelCode Blue Stain Reagent (Thermo Fisher Scientific) for Coomassie brilliant blue staining. citrulline content (= 6). Each sign represents a single mouse. The horizontal and vertical bars represent the mean and SEM values for the group, respectively. (TIFF 149 kb) 13075_2018_1562_MOESM3_ESM.tif (149K) GUID:?8C920EFC-5DDA-4BBF-9C88-0E233B00BBD5 Additional file 4: Figure S4. BAY-598 (a) Sera of pGIA and control mice obtained at day 14 were separated by SDS-PAGE and stained with Coomassie amazing blue (= 60, mean age 52.2 years, range 20C73 years, females 80%) diagnosed by rheumatologists according to the 1987 American College of Rheumatology (ACR) classification criteria [14] or the 2010 ACR/European League Against Rheumatism classification criteria [15]. Serum samples of the disease control subjects were collected from Japanese patients with main Sj?grens syndrome (SS) (= 27, mean age 58.8 years, range 26C80 years, females 96%), systemic lupus erythematosus (SLE) (= BAY-598 15, mean age 33.9 years, range 16C55 years, KRT17 females 80%), or osteoarthritis (OA) (= 12, mean age 56.3 years, range 37C80 years, females 67%). All the patients with SS were diagnosed by rheumatologists according to the 1999 Japanese Ministry of Health criteria for diagnosis of SS [16]. All the patients with SLE fulfilled the 1997 ACR classification criteria [17]. None of the patients with SS or SLE experienced overlapping RA. Serum samples were collected from healthy subjects (HS) (= 30, mean age 49.0 years, range 34C65 years, females 80%). Serum samples were also collected from 17 patients with RA before and 24 weeks after treatment with biologic drugs (infliximab, = 9; abatacept, = 8). All samples were collected at the University or college of Tsukuba Hospital after knowledgeable consent was obtained. This study was examined and approved by the ethics committee of the University or college of Tsukuba. Peptide GPI-induced arthritis DBA/1 mice were immunized with 25 g of pGPI (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA) in total Freunds adjuvant (CFA) (BD Biosciences, San Jose, CA, USA). pGPI was emulsified with CFA at a 1:1 ratio (vol/vol), or PBS + CFA was prepared as a vehicle control. For induction of arthritis, 150 l of the emulsion was injected intradermally at the base of the tails of the mice. Each mouse was also given an injection of 200 ng of pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally on days 0 and 2 after immunization to induce arthritis. Arthritis was assessed every other day and evaluated using a level of 0C3 for swelling and redness of each paw. The clinical score was the sum of the scores for four paws, as described previously [11]. Measurement of anti-CCP antibodies in pGIA Sera were obtained on days 0C28 every week from mice immunized with pGPI or control, and antibodies were measured by enzyme-linked immunosorbent assay BAY-598 (ELISA). Sera were diluted 1:25 in dilution buffer and added to the 96-well plate (Immunoscan CCPlus test kit; Euro Diagnostica, Malm?, Sweden) for 1 h at room heat. After a washing step, horseradish peroxidase (HRP)-conjugated polyclonal rabbit antimouse immunoglobulin (Dako, Carpinteria, CA, USA) diluted 1:1000 was added for 30 minutes at room heat. After another washing step, color was developed with 3,3,5,5-tetramethylbenzidine (TMB) microwell peroxidase substrate (KPL/SeraCare, Milford, MA, USA). The optical density (OD) was measured at 450 nm by using a microplate reader. A standard pool was obtained by mixing sera obtained from several mice on day 28. The concentrations of antibodies in this pool were considered 100 U/ml. A standard curve was obtained using serum dilutions, and the Michaelis-Menten equation was used to convert OD values into units, as described previously [18]. Measurement of anti-ITIH4 antibodies in patients with RA Native peptide ITIH4428C447 and R438 citrullinated peptide ITIH4428C447 were synthesized (serum, purity 95%) and used in ELISA. Ninety-six-well plates (Nunc MaxiSorp; Thermo Fisher Scientific) were coated with 10 g/ml peptides for 12 h at 4 C. After washing and blocking actions, sera from patients with RA (= 60) and HS (= 30) were diluted 1:200 in 1% bovine serum albumin (BSA) in PBS, then added for 2 h at room heat. After a washing step, HRP-conjugated goat antihuman immunoglobulin G (IgG) (heavy and light chains [H + L]) (Abcam, Cambridge, MA, USA) diluted 1:10,000 was added for 1 h at room temperature. After washing, color.