Anti-β₂GPI peptide Abs bind human TLR4. (A) Crystal structure of TLR4. The sequence of the PDDLPFST peptide is highlighted (white). (B) Immunoreactivity of anti-β₂GPI peptide Abs to TLR4 was analyzed by Western blot. Cell lysates from HEK293T4 cells were probed with human purified anti-BSA IgG (lane 1), rabbit anti–human TLR4 Abs (lane 2), and human purified anti-β₂GPI peptide IgG (lane 3), followed by HRP-linked goat anti–human or anti–rabbit IgG Abs. Immunoreactivity was assessed by ECL. (C) Fluorescent confocal microscopy images showing a coimmunolocalization of TLR4 (green) and β₂GPI peptide (red) on the HEK293T4 cell surface using monoclonal anti-TLR4–FITC conjugated and human purified anti-β₂GPI peptide Abs (0.1 μg/μL), followed by Texas Red–conjugated anti–human IgG Abs. Merged image (overlay) depicts areas of overlap in yellow. (D) Fluorescent confocal microscopy images of untransfected HEK293 cells as a negative control. (E) HEK293T4 cells were immunoprecipitated with human anti-β₂GPI peptide Abs. The immunoprecipitates were analyzed by Western blotting using polyclonal anti-TLR4 Abs. Bound Abs were visualized with HRP-conjugated anti–rabbit IgG and immunoreactivity was assessed by ECL. Virtually, no reactivity was found with immunoprecipitates obtained using control irrelevant IgG.