Involvement of the PAR site in the distal promoter of LMO2 gene in E2A-HLF–induced LMO2 expression. (A) Schematic representation of the proline and acidic amino acid–rich protein (PAR) site in the distal promoter of the LMO2 gene and oligonucleotides used as competitors in electrophoretic mobility shift assay. Mutations in the sequence are underlined. (B) Electrophoretic mobility shift assay performed in nuclear extracts from UOC-B1 cells using an HLF-CS sequence as a probe in the presence of a series of double-stranded oligomers as competitors (lanes 4-12) or anti-HLF(C) (lane 1) and anti-E2A (lane 2) sera. The molar ratio of cold competitor to probe is indicated in each lane. The specific DNA-protein complex is indicated by the bracket and the supershifted complex is indicated by the arrowhead. (C) Schematic representation of 3 reporter constructs for reporter assay. Mutations in the sequence are underlined. (D) Western blot analysis of E2A-HLF–transfected Nalm6 cells. Lysates of UOC-B1 cells and wild type and E2A-HLF transfected-Nalm6 cells cultured in the absence or presence of zinc for 24 hours were blotted with E2A antisera. Open and closed arrowheads indicate E2A and E2A-HLF, respectively. (E) Luciferase assay in E2A-HLF–transfected Nalm6 cells. Assays were performed in wild type and E2A-HLF–transfected Nalm6 cells cultured in the absence or presence of zinc for 24 hours after transient transfection of each reporter plasmid. The values were normalized for transfection efficiencies using a cotransfected Renilla luciferase construct. (F) Luciferase assay in YCUB2 cells. The values were normalized for transfection efficiencies using a cotransfected Renilla luciferase construct.