Detection of annexin A2 and TLR4-associated proteins by immunoprecipitation. ECs were either untreated or incubated with β₂GPI and anti-β₂GPI Abs for 5 hours. Detergent extracts were prepared and analyzed by 4%-12% SDS-PAGE before (A) or after immunoprecipitation using Abs against nucleolin (B), annexin A2 (C), or TLR4 (D). Whole extracts and each of the immunoprecipitates were then immunoblotted using Abs against annexin A2, nucleolin, calreticulin, or TLR4. Extracts were also immunoprecipitated using control IgG, which did not precipitate any detectable protein. The results shown are from 1 representative experiment of 3.