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Additional file 1: Figure 1. Sequence, structure and major immunogenic region (mir) of hnRNP-D and hnRNP-DL. A, Schematic representation of hnRNP-D (isoform p45), hnRNP-DL and the different recombinant hnRNP-DL variants studied. The main structural features are highlighted. Mir-region is the major immunogenic region, RBD1 and RBD2 are RNA-binding domains 1 and 2, Gly-rich is the C-terminal glycine-rich region of the proteins. B, Global amino acid sequence alignment of hnRNP-D and hnRNP-DL1 (isoform 1). HnRNP-D and -DL share 89.1% similarity by sequenc e[1]. Regions “mir”, “RBD1”, “RBD2” and “Gly-rich” are highlighted. Figure 2. Characterisation of autoantibodies against, A, citrullinated α-hnRNP-DLmir (cit-DL), B, α-hnRNP-DLmir (DL) and C, ∆OD between cit-DL and DL (ΔDL) determed by ELISA in sera of other diseases (n=127; MS n=20, reA n=7, Sclero n=20, Sjö n=20, PsA n=20, MB n=20, OA n=20). The dotted lines markes the cutoff vs. other diseases (except systemic lupus erythematosus) or healthy controls with 98% specificity each. OD, optical density; nm, nano meter; vs., versus; MS, multiple sclerosis; reA, reactive arthritis; Sclero, scleroderma; Sjö, Sjögren´s syndrome; PsA, psoriasis arthritis; MB, ankylosing spondylitis; OA. Osteoarthritis. Table 1. Mann Whitney U-test of (cit) α-hnRNP-DLmir-OD signals of seropositive and seronegative data sets of RA-cohorts. Table 2. Mann Whitney U-test of cit α-hnRNP-DLmir-OD signals of seronegative data sets of RA-cohorts and data sets of other inflammatory diseases. Figure 3. XY-Plot and Spearman Correlation of citrullinated or native α-hnRNP-DLmir versus ΔhnRNP-DLmir for the early RA cohort EIRA (A/D; n=404), the seropositive EIRA sera (B/E; n=202) and the seronegative EIRA sera (C/F; n=202). Table 3. Spearman correlation of the early RA sera of the EIRA cohort (n=404). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 4. Spearman correlation of the 242 EIRA sera treated with MTX (α-CCP2 positive n=133, α-CCP2 negative n=109). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 5. Spearman correlation of the established RA sera of the Predict cohort (n=94; RF IgM and/or α-CCP2 positive n=64, RF IgM and α-CCP2 negative n=30). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 6. ROC analysis of native hnRNP-DLmir of MTX-treated EIRA patients (n=192; seropositive n=93, seronegative n=99). Table 7. Negative CNDL-index of MTX-treated EIRA patients n=192 (Resp. n=161, non-Resp. n=31). Table 8. ROC analysis of native hnRNP-DLmir of Enbrel®-treated Predict patients (n=94; seropositive n=63, seronegative n=31). Figure 4. High baseline titer against α-hnRNP-DLmir (DL) is rather present in 6-month EULAR Responder RA patients who had received MTX or α-TNF inhibitor therapy (Enbrel®). A-C, Citrullinated α-hnRNP-DLmir (citDL) (A), α-hnRNP-DLmir (DL) (B) and ∆ OD between citDL and DL (ΔDL) (C) were measured by ELISA in patient sera from the EIRA cohort treated with MTX (n=192) with 161 EULAR Responder and 31 EULAR non-Responder among 6 months. The evaluation was done according to the cutoff versus other diseases. D, α-DL were measured by ELISA in patient sera from the Predict cohort treated with α-TNF inhibitor therapy with 6-month EULAR response data (n=94, responder n=63, non-Responder n=31). Based on the signals, a response-cutoff (dotted line, OD 0.174) was determined, from which only responders are recognized as positive. OD, optical density; nm, nano meter; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; MTX, Methotrexate; Resp., 6-month EULAR Responder. Figure 5. A, Influence of cytokines on hnRNP-DL expression determined by immunoblotting. Cellular extracts from unstimulated, IL1α- or TNFα-stimulated HeLa cells and from unstimulated and IL6-stimulated HepG2 cells were probed with α-hnRNP-DL1/2-peptide specific rabbit serum. B, Citrullination of hnRNP-DL in synovial tissue from a patient with rheumatoid arthritis was investigated with an α-deiminated arginine antibody and an α-hnRNP-DL antibody. Both positive bands were labled with hnRNP-DL, which isoforms were not analysed.

Additional file 1: Figure 1. Sequence, structure and major immunogenic region (mir) of hnRNP-D and hnRNP-DL. A, Schematic representation of hnRNP-D (isoform p45), hnRNP-DL and the different recombinant hnRNP-DL variants studied. The main structural features are highlighted. Mir-region is the major immunogenic region, RBD1 and RBD2 are RNA-binding domains 1 and 2, Gly-rich is the C-terminal glycine-rich region of the proteins. B, Global amino acid sequence alignment of hnRNP-D and hnRNP-DL1 (isoform 1). HnRNP-D and -DL share 89.1% similarity by sequenc e[1]. Regions “mir”, “RBD1”, “RBD2” and “Gly-rich” are highlighted. Figure 2. Characterisation of autoantibodies against, A, citrullinated α-hnRNP-DLmir (cit-DL), B, α-hnRNP-DLmir (DL) and C, ∆OD between cit-DL and DL (ΔDL) determed by ELISA in sera of other diseases (n=127; MS n=20, reA n=7, Sclero n=20, Sjö n=20, PsA n=20, MB n=20, OA n=20). The dotted lines markes the cutoff vs. other diseases (except systemic lupus erythematosus) or healthy controls with 98% specificity each. OD, optical density; nm, nano meter; vs., versus; MS, multiple sclerosis; reA, reactive arthritis; Sclero, scleroderma; Sjö, Sjögren´s syndrome; PsA, psoriasis arthritis; MB, ankylosing spondylitis; OA. Osteoarthritis. Table 1. Mann Whitney U-test of (cit) α-hnRNP-DLmir-OD signals of seropositive and seronegative data sets of RA-cohorts. Table 2. Mann Whitney U-test of cit α-hnRNP-DLmir-OD signals of seronegative data sets of RA-cohorts and data sets of other inflammatory diseases. Figure 3. XY-Plot and Spearman Correlation of citrullinated or native α-hnRNP-DLmir versus ΔhnRNP-DLmir for the early RA cohort EIRA (A/D; n=404), the seropositive EIRA sera (B/E; n=202) and the seronegative EIRA sera (C/F; n=202). Table 3. Spearman correlation of the early RA sera of the EIRA cohort (n=404). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 4. Spearman correlation of the 242 EIRA sera treated with MTX (α-CCP2 positive n=133, α-CCP2 negative n=109). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 5. Spearman correlation of the established RA sera of the Predict cohort (n=94; RF IgM and/or α-CCP2 positive n=64, RF IgM and α-CCP2 negative n=30). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 6. ROC analysis of native hnRNP-DLmir of MTX-treated EIRA patients (n=192; seropositive n=93, seronegative n=99). Table 7. Negative CNDL-index of MTX-treated EIRA patients n=192 (Resp. n=161, non-Resp. n=31). Table 8. ROC analysis of native hnRNP-DLmir of Enbrel®-treated Predict patients (n=94; seropositive n=63, seronegative n=31). Figure 4. High baseline titer against α-hnRNP-DLmir (DL) is rather present in 6-month EULAR Responder RA patients who had received MTX or α-TNF inhibitor therapy (Enbrel®). A-C, Citrullinated α-hnRNP-DLmir (citDL) (A), α-hnRNP-DLmir (DL) (B) and ∆ OD between citDL and DL (ΔDL) (C) were measured by ELISA in patient sera from the EIRA cohort treated with MTX (n=192) with 161 EULAR Responder and 31 EULAR non-Responder among 6 months. The evaluation was done according to the cutoff versus other diseases. D, α-DL were measured by ELISA in patient sera from the Predict cohort treated with α-TNF inhibitor therapy with 6-month EULAR response data (n=94, responder n=63, non-Responder n=31). Based on the signals, a response-cutoff (dotted line, OD 0.174) was determined, from which only responders are recognized as positive. OD, optical density; nm, nano meter; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; MTX, Methotrexate; Resp., 6-month EULAR Responder. Figure 5. A, Influence of cytokines on hnRNP-DL expression determined by immunoblotting. Cellular extracts from unstimulated, IL1α- or TNFα-stimulated HeLa cells and from unstimulated and IL6-stimulated HepG2 cells were probed with α-hnRNP-DL1/2-peptide specific rabbit serum. B, Citrullination of hnRNP-DL in synovial tissue from a patient with rheumatoid arthritis was investigated with an α-deiminated arginine antibody and an α-hnRNP-DL antibody. Both positive bands were labled with hnRNP-DL, which isoforms were not analysed.