Macrophage migration inhibitory factor (MIF) activates c-jun N-terminal kinase (JNK) and c-jun in rheumatoid arthritis (RA) synovial fibroblasts. (a–c) RA synovial fibroblasts were stimulated with MIF (25 nM) for 1 minutes, 5 minutes, 15 minutes, 30 minutes, and 45 minutes. Phosphorylated JNK and c-jun signaling molecule expression in cell lysates was measured by western blot. Blots were stripped and re-probed with antiactin antibody to verify equal loading. MIF-induced phosphorylation of JNK (a) and c-jun (b). Results represent one of four similar experiments using cells from four donors. (c–d) Immunofluorescence staining of RA synovial fibroblasts shows the expression of different phospho-signaling molecules in the presence or absence of MIF (50 nM) for 25 minutes and with or without DAPI nuclear staining. MIF-induced diffuse cytoplasmic upregulation of *p-JNK (c), increased the nuclear expression of *p-c-jun (d) (400×). DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; NS, nonstimulated; *p-JNK, phospho-c-jun N-terminal kinase; rhMIF, recombinant human macrophage migration inhibitory factor; *p-c-jun, phospho-c-jun.