IRAK and gp130 contribute to the modulation of EC signaling and E-selectin expression by SMC-coculture and shear stress. (A) ECs were kept as controls or transfected with control siRNA (siCL) or a specific siRNA of gp130 (sigp130) or IRAK (siIRAK) at indicated concentrations for 48 hours, and their gp130 or IRAK mRNA expression was determined by RT-PCR analysis, as described in “Materials and methods.” (B-E) ECs were kept as controls (EC/∅) or cocultured with SMCs in the adjacent-bilayer model (EC/SMC) for 10 minutes (E), 4 hours (B-C), or indicated times (D). Before coculture with SMCs, ECs were transfected with control siRNA or a specific siRNA of gp130 (B) or IRAK (C) (100 μmol/mL for each) for 48 hours or were presheared at HSS (HS) or LSS (LS) for 24 hours (E). Control ECs (CL) were cocultured with SMCs without transfection (B-C) or preshearing (E). The mRNA expressions of E-selectin and gp130 (B) or IRAK (C), or the phosphorylations of gp130 and IRAK (D-E) in these ECs were determined by using RT-PCR analysis or immunoprecipitation assay and Western blot analysis, respectively. Results in (A-E) are representative of triplicate experiments with similar results.