Affinity-purified antivimentin/cardiolipin antibodies from SN-APS induce IRAK1 phosphorylation and activate NF-κB. Endothelial cells were stimulated with affinity-purified antivimentin/cardiolipin antibodies (200 μg/mL), NHS-IgG fraction (200 μg/mL), LPS (100 ng/mL), and affinity-purified antivimentin antibodies (200 μg/mL) for 45 minutes at 37°C, in 5% CO₂ and cellular extracts were obtained. (A) Phosphorylated levels of IRAK1 (p-IRAK1) were analyzed in whole-cell extracts by Western blotting with antiphospho-IRAK1 antibodies; for control, the blotted membranes were stripped and reprobed with antiactin antibodies. Bound antibodies were visualized with horseradish peroxidase-conjugated IgG and immunoreactivity was assessed by ECL. One example representative of the patients with antivimentin/cardiolipin antibodies. (B) NF-κB activation was analyzed in nuclear cell extracts by Western blot with antiphospho–NF-κB p65 Ser antibodies; for a control, the blotted membranes were stripped and reprobed with anti–histone H1 antibodies. Bound antibodies were visualized with horseradish peroxidase-conjugated IgG, and immunoreactivity was assessed by ECL. One example representative of the patients with antivimentin/cardiolipin antibodies is shown.