Effects of TLR4 knockdown. (A) Flow cytometric analysis of cell surface TLR4 expression 48 hours after siRNA transfection (left panel: not transfected cells; right panel: AllStars negative control siRNA- and TLR4 siRNA-transfected cells). The dotted line represents isotype control; the black line represents TLR4 expression in nontransfected and TLR4 siRNA-transfected cells; the gray line represents TLR4 expression in negative control transfected cells (AllStars siRNA). A representative experiment of 3 is shown. (B) IRAK phosphorylation was evaluated by Western blot analysis. THP1 cells (silenced with TLR4 siRNA and control siRNA) were treated for 45 minutes with human purified endotoxin-free anti-β2GPI peptide and, as controls, with anti-BSA Abs (100 μg/mL); or with E coli LPS (100 ng/mL). Cell lysates were probed with anti–phospho-IRAK1 and anti–α-actin polyclonal Abs as a loading control. Bound Abs were visualized with HRP-linked goat anti–rabbit IgG and immunoreactivity was assayed by ECL. Scanning densitometric analysis of each sample is shown. (C) NF-κB activation was analyzed by treating THP1 cells (silenced with TLR4 siRNA and control siRNA) for 45 minutes with human purified endotoxin-free anti-β2GPI peptide and, as controls, with anti-BSA Abs (100 μg/mL) or with E coli LPS (100 ng/mL). Equal amounts of whole-cell lysates were used to determine the levels of the activated p65 subunit using Abs directed against the subunit bound to the oligonucleotide containing the NF-κB consensus-binding site. Values are the means ± SDs of 3 different experiments.
