Anti-β₂GPI peptide Abs activate monocytes. (A) IRAK phosphorylation was evaluated by Western blot. Monocytes were treated for 45 minutes with human purified endotoxin-free anti-β₂GPI peptide and, as controls, with anti-BSA Abs (100 μg/mL); with E coli LPS (100 ng/mL); with purified IgG from an APS patient positive for the β₂GPI peptide (200 μg/mL); or with APS IgG (200 μg/mL) preabsorbed with β₂GPI peptide. Whole-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. (B) NF-κB activation was analyzed by treating monocytes for 45 minutes with human purified endotoxin-free anti-β₂GPI peptide and, as controls, with anti-BSA Abs (100 μg/mL); with E coli LPS (100 ng/mL); with APS IgG (200 μg/mL); or with APS IgG (200 μg/mL) preabsorbed with β₂GPI peptide. 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. Statistical analysis was performed by the Student paired t test (P < .001). * indicate versus control. (C) Release of TNF-α was analyzed using a commercially available ELISA kit. Cells were stimulated for 48 hours with human purified endotoxin-free anti-BSA and anti-β₂GPI peptide Abs (100 μg/mL) and E coli LPS (100 ng/mL) and, after the treatment, supernatants were collected and analyzed. Values are the means ± SDs of 3 different experiments. Statistical analysis was performed by the Student paired t test (P < .001). * indicates versus control.