NF-κB is involved in SMC and shear stress modulations of E-selectin expression in ECs. ECs were kept as controls (EC/∅) or cocultured with SMCs in the adjacent-bilayer model (EC/SMC) for 30 minutes (D-G,I), 24 hours (A), or indicated times (B,C,H). Before kept as controls or coculture with SMCs, ECs were (1) transfected with E-selectin–Luc for 48 hours and/or pretreated with antisense (p65-a; 1 μg/mL) or sense (p65-s; 1 μg/mL) oligonucleotides to the NF-κB subunit p65 for 24 hours or with lactacystin (Lac; 20 μM) or N-acetyl-cysteine (NAC; 20 mM) for 1 hour (A), (2) presheared at HSS (HS) or LSS (LS) for 24 hours (D,F,I), or (3) pretreated with SP600125 (SP; 20 μM) or SB203580 (SB; 10 μM) individually or in combination (CB) for 1 hour (E,F,I). Control ECs (CL) were cocultured with SMCs without any preshearing (D,F,I) or pretreatment (D,F,I). The E-selectin promoter activity (A), NF-κB–DNA binding activity (B,D,E,G), IκBα protein expression (C,F), and in vivo NF-κB–promoter binding (H-I) in these ECs were determined by using luciferase assay (A), EMSA (B,D,E,G), Western blot analysis (C,F), and ChIP assay (H,I), respectively, as described in “Materials and methods.” In some experiments (G), EMSA was performed using total nuclear extracts and ³²P-labeled oligonucleotides containing wild-type (CL) or mutant (Mut) human E-selectin NF-κB binding sites. The specificity of the retarded complexes (NF-κB) was assessed by preincubating the nuclear extracts either with 20-fold excess unlabeled oligonucleotides (wild-type or mutant) as a competitor or with p50 and/or p65 antibodies (1 μg). Nuclear extracts preincubated with the p65 antibody show a super shift band (SH) (G). (B-G) The results are representative of 2 or 3 independent experiments with similar results. (A,H-I) Data are represented as mean ± SEM from 3 to 5 independent experiments. *P < .05 versus control EC/∅. ^#P < .05 versus control EC/SMC.