Testing phosphoSer172 IKKe antibody (S824C). RAW264.7 macrophages were either left unstimulated (-) or stimulated (+) with 100 ng/ml LPS for 30 min. Cell extracts (0.5 mg) were incubated with 5 mg of each antibody in the presence of 50 mg of unphosphorylated form of the peptide immunogen with (+) or without (-) the addition of 50 mg of phosphopeptide. Immunoprecipitated proteins were resolved by SDS-PAGE on a 8% gel and immunoblotted using antibodies that recognize all forms of TBK1 and IKKe that were purchased from Cell Signaling Technology. Note that bleed 3 gives similar results to the previously used antibody raised in S051c. Immunoprecipitation: This antibody immunoprecipitates TBK1 and IKKepsilon phosphorylated on Ser172. The immunoprecipitation of these proteins can be blocked using the phosphopeptide antigen. Note that the immunoprecipitated proteins are detected using antibodies raised against total TBK1 and IKKepsilon which are purchased from Cell Signaling Technology.