HMGB2 is associated with oligonucleotide corresponding to the GFI1B promoter. (A) Proteins bound to GFI1B promoter oligonucleotide were separated by SDS-PAGE on a 10% polyacrylamide gel. After Coomassie staining, the protein band with a 25-kDa apparent molecular weight was cut and analyzed by mass spectrometry. (B) Extracted peptides were analyzed by mass spectroscopy using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Voyager DEPro; Applied Biosystems). Monoisotopic mass list was used to search the Swiss-Prot database²⁷  for human proteins using the Mascot search engine. (C) Schematic representation of the GFI1B promoter. Two specific regulatory regions were indicated “site 1” (−138 to −116) and “site 2” (−69 to −37). The 2 putative Oct-1–binding sites identified in this study and the NF-Y–binding site were indicated. (D) GATA-1, Oct-1, NF-Y, and HMGB2 association with oligonucleotides corresponding to the GFI1B promoter. Oligo pull-down assays were performed with cell lysates from differentiated erythroid cells harvested 3 days after induction of erythroid differentiation (E3) of CD34⁺ cells. Cell lysates were immunoprecipitated with increasing amount of oligonucleotides representing wild-type (indicated above the lanes by black wedges) or mutated site 1 or site 2 of the GFI1B promoter. Proteins bound to the DNA template were separated by SDS-PAGE and analyzed by Western blotting using antibodies against GATA-1, Oct-1, NF-YA, or HMGB2. The results shown on this figure are representative of 4 experiments.