Methotrexate (MTX) treatment of rheumatoid arthritis (RA) patients ( n = 11) does not modulate synovial expression of either citrullinated proteins or PAD2 and PAD4 enzymes. Hematoxylin-eosin (HTX-EOS) staining illustrates the histologic pattern of frozen synovial biopsy sections of an RA patient before (a) and after (b) MTX treatment. Brown diaminobenzidine immunoperoxidase staining shows no changes in expression of citrullinated proteins as detected by F95 antibody (c, before treatment; d, after treatment), PAD2 as detected with either ROI001 antibody (f, before treatment; g, after treatment), or SN665 antibody (i, before treatment; j, after treatment) and PAD4 as detected with either SN823 antibody (l, before treatment; m, after treatment), or SG1467 antibody (o, before treatment; p, after treatment). Original magnification ×100, whereas insets show close-up details from the same samples. Graphs show results of semiquantitative analysis on a 4-grade scale for citrullinated proteins (e), PAD2 as detected with either ROI001 (h) or SN665 (k) antibody, and PAD4 as detected with either SN823 (n) or SG1467 (q) antibody. Values represent the median, and whiskers, the range (*P < 0.05).