Figure 3.

H. influenzae induces NF-κB binding to the ICAM-1 gene. (A) Transcription factor binding to ICAM-1 HFRE gene sequence from −200 to −135 was assessed using electrophoretic mobility shift assay (EMSA) with nuclear extracts from hTBE cells that were incubated without or with H. influenzae for 4 hours. Specificity of protein binding was assessed by competition with unlabeled oligonucleotides containing the HFRE, but not an unrelated CRE. Protein components of the DNA-binding complexes were identified using supershift analysis by addition of antibodies against p65, p50, or control. The position of ICAM-1 HFRE-binding complexes is indicated by arrows. (B) Transcription factor binding to the CRE was assessed using EMSA with the same nuclear extracts as in A to verify equivalency of nuclear protein isolation. The position of CRE-binding complexes is indicated by an arrow. Results in both panels are representative of five experiments.