Transcription factor binding to the κB sites in the CCR5 downstream promoter. EMSA showing binding of complexes to the κB sites of CCR5 (κB-1 through -3) and the κB site of human β2m as a consensus probe (cons) using nuclear extracts of THP-1 cells (left panels) or U251 cells (right panels). (A) Cells were left untreated or stimulated with TNF-α (10 ng/mL) for 2 hours. EMSA analysis revealed formation of 2 complexes to CCR5-κB-1 on TNF-α treatment (→). CCR-5-kB-2 showed nonspecific complex binding constitutively, whereas CCR-5-κB-3 did not generate any significant binding, either constitutive or on TNF-α treatment. (B) Using specific antibodies, the proteins binding to CCR5-κB-1 on TNF-α treatment were identified as NF-κB p65 and p50. Binding of these factors to CCR5-κB-2 could not be detected. *Supershifted complexes. Shown are representatives of 2 independent experiments.