Activation of the TLR4/MyD88/NF-κB signaling pathway is dependent on assembly of the multiprotein signaling complex. (A) ECs were plated in 96-well plates and either not pretreated or pretreated with a cell-permeable peptide that contains a sequence from the MyD88/TIR homodimerization domain that blocks MyD88-dependent TLR4 signaling. Cells were then exposed to LPS, β₂GPI and anti-β₂GPI Abs, or TNFα. The peptide inhibited EC activation in response to LPS and anti-β₂GPI Abs, but not TNFα, demonstrating the dependence of activation on a TLR4/MyD88 pathway.¹⁹  Error bars represent the means ± SD of triplicate points. *P < .0001 for cells treated with β₂GPI/anti-β₂GPI Abs alone versus β₂GPI/anti-β₂GPI in the presence of the homodimerization domain peptide. (B) ECs were pretreated with control RNA of random sequence but identical composition as specific siRNA (lanes 1 and 2), TLR4 siRNA (lane 3), annexin A2 siRNA (lane 4), calreticulin siRNA (lane 5), nucleolin siRNA (lane 6), or all 4 siRNAs simultaneously (lane 7). Cells were then incubated with β₂GPI and anti-β₂GPI Abs, and cell lysates prepared and assessed for phosphorylated NF-κB p65 (S536) and total NF-κB content by immunoblotting. (C). ECs were transfected with a construct containing an NF-κB promoter sequence linked to a luciferase reporter gene. Cells were then treated with control RNA or siRNA directed against annexin A2, TLR4, nucleolin, or calreticulin. After incubation with β₂GPI and anti-β₂GPI Abs, cell lysates were prepared and luciferase activity determined. Pretreatment of cells with each of the siRNAs caused significant reductions in luciferase activity compared with cells pretreated with control RNA (P < .0001 by ANOVA). Error bars represent the means ± SD of triplicate points. The results shown are from 1 representative experiment of 3.