Figure 1. The human MIF gene and the strategy for the identification of MIF –794 CATT_5–8–interacting proteins.

(A) Diagram illustrating the 3 MIF exons, predicted transcription factor–binding sites, and the –794 CATT_5–8 microsatellite repeat (rs5844572). The numerical prefixes refer to nucleotide distance (in bp) upstream from the transcription start site. (B) Diagram of synthetic 5′ biotin-labeled oligonucleotides spanning nucleotides –865/–833 to –752 of the MIF promoter used for differential affinity chromatography of human THP-1 monocyte nuclear proteins. The corresponding double-stranded oligos were used experimentally. (C) Verification of retention of the nuclear transcription factor Pit-1 by the 5′CATT₈ but not 5′CATT₀ oligonucleotide. Recombinant Pit-1 (10 ng) incubated with 100 nM MIF promoter 5′CATT₀ or 5′CATT₈ oligos prior to the addition of streptavidin beads, NaCl elution, and SDS-PAGE (4%–12%) of eluates, followed by Western blotting with anti–Pit-1 (lanes 1 and 2). Positive control Western blot showing recombinant Pit-1 alone (no oligonucleotide addition) (lane 3). The blot is representative of 3 experiments.