Fig. 1.

4-Hydroxy-2-nonenal (HNE) modification of proteins was validated using IP and Western blotting (a). Proteins were immunoprecipitated from pooled concanavalin (Con) A-captured serum samples (32 healthy controls (HCs) and 32 patients with rheumatoid arthritis (RA)) using anti-complement factor H (CFAH), anti-haptoglobin (HPT), anti-immunoglobulin kappa chain C region (IGKC), and anti-prothrombin (THRB) antibodies and then subjected to Western blotting with anti-HNE antibodies (upper panel). Individually selected random serum samples (HCs and RA patients) were used as controls; these were simultaneously used for Western blotting with anti-HNE antibodies. Percentages of the SDS-PAGE gel and IP loading amounts of Con A-captured serum proteins were 8% and 20 µg, 10% and 20 µg, 12% and 5 µg, and 8% and 20 µg for CFAH, HPT, IGKC, and THRB, respectively. A duplicate gel was stained with Coomassie brilliant blue (CBB) as a loading control, including CFAH (b), HPT (c), IGKC (d), and THRB (e). Red arrows indicate immunoprecipitated proteins