(A) 2D gel electrophoresis and silver staining of human THP-1 monocyte nuclear proteins eluted from the MIF promoter 5′CATT₀- (control), 5′CATT₅-, and 5′CATT₈-containing oligonucleotides, respectively. (B) MIF promoter 5′CATT_X length–dependent retention of ICBP90 detected by Western blotting of eluted nuclear proteins with an anti-ICBP90 antibody. Anti–β-actin served as a protein loading control. (C) EMSA verification of the interaction of ICBP90 with the 5′CATT₈ site in the MIF promoter. Nuclear lysate decreases the mobility of a biotin-labeled, CATT₈-containing MIF promoter oligonucleotide (lanes 1 and 6). This effect is eliminated by the addition of a 200-fold excess of unlabeled 5′CATT₈ oligonucleotide (lane 3) but not 5′CATT₀ oligonucleotide (lane 2) and inhibited by anti-ICBP90 (lane 4) but not control IgG (lane 5). (D) Amplification of MIF promoter DNA by ChIP of human PBMC DNA with anti-ICBP90 but not control IgG. RU, fluorescence relative units of amplified DNA. Similar results were obtained with PBMC DNA from healthy control and subjects with rheumatoid arthritis (data not shown). (E) ICBP90 mRNA expression analyzed by qPCR in 1 × 10⁶ HEK293 cells transfected 16 hours previously with pCMV-ICBP90 or an empty vector (pCMVcon). (F) Intracellular ICBP90 protein analyzed by Western blotting of cell lysates together with a β-actin loading control. (G) Corresponding changes in intracellular MIF mRNA (qPCR), (H) intracellular MIF protein content (Western blotting), and (I) MIF secretion into 24-hour conditioned medium (ELISA). Data are mean+SD of 3 measurements, with all experiments replicated twice (n = 3 measurements per experiment). **P < 0.01 by 2-tailed Student’s t test. Displayed blots are representative of 3 independent experiments.