IL-1β and IL-6 produced by EC/SMC are the major factors contributing to the SMC-induced signaling and E-selectin expression in ECs. (A) Detection of protein levels of cytokines in conditioned media of EC/EC or EC/SMC coculture. The membranes spotted with antibodies against 120 different cytokines and other proteins (Figure S2) were incubated with 2-fold–diluted conditioned media of EC/EC or EC/SMC coculture and then incubated with a mixture of biotin-labeled antibodies, as described in “Materials and methods.” Signal detection by enhanced chemiluminescence (ECL) shows that the expression levels of IL-1β (spots in solid boxes) and IL-6 (spots in dash boxes) produced by EC/SMC were significantly higher than that of EC/EC. Results are representative of 4 independent experiments with similar results. (B-F) ECs were kept as controls (EC/∅) or cocultured with SMCs in the adjacent-bilayer model (EC/SMC) for 30 minutes (C-F) or 4 hours (B). In parallel experiments, ECs were preincubated with a neutralizing antibody against IL-1β or IL-6 (5 μg/mL for each) or their combination for 1 hour and then cocultured with SMCs in the presence of the antibodies. Control ECs were cocultured with SMCs in the presence of control IgG (CL). The E-selectin mRNA expression (B), JNK and p38 phosphorylations (C), NF-κB-DNA binding activity (D), IκBα protein expression (E), and in vivo NF-κB–promoter binding (F) were determined by using Northern blot analysis, Western blot analysis, EMSA, and ChIP assay, respectively, as described in “Materials and methods.” (B-C,E) Data are presented as percentage changes in band densities from control EC/∅ normalized to GAPDH RNA (B), JNK or p38 protein (C), or actin protein (E). The results shown are mean ± SEM from 3 to 4 independent experiments. *P < .05 versus control EC/∅. ^#P < .05 versus control EC/SMC.