Oxidized β₂-GPI induces IRAK phosphorylation and activates NFκB. DCs were stimulated with ox-β₂-GPI, H₂O₂-β₂-GPI, αTOC-β₂-GPI, or LPS, or left unstimulated for 45 minutes. Cellular extracts were obtained. (A) Phosphorylated levels of IRAK (p-IRAK) were analyzed by Western blotting with antiphosphoserine mAb. Bound antibodies were visualized with HRP-conjugated anti-mouse IgG and immunoreactivity was assessed by ECL. Immunoprecipitates obtained using unstimulated cells, non-IRAK-specific IgG (irrelevant), and αTOC-β₂-GPI-stimulated cells yielded only a weak band. The figure shows a representative experiment from 3 with similar results. (B-C) After cell lysing, protein was quantified, and equal amounts of lysates were used to test activated levels of the p50 and p65 subunits with antibodies directed against the subunits bound to the oligonucleotide containing the NFκB consensus binding site. An HeLa cell extract was used as a positive control alone or in the presence of wild-type or mutated consensus oligonucleotide. Results are expressed as mean ± SD from 3 different experiments. Significant differences between unstimulated samples and samples stimulated with LPS, H₂O₂-β₂-GPI, and ox-β₂-GPI are indicated with asterisks (p50: ^*P = .013, ^**P = .065, and ^***P = .05; p65: ^*P < .001, ^**P = .006, and ^***P = .007).