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Peritoneal macrophages were untreated or stimulated with Pam3CSK4 for 30 min. Cells were collected and lysed in cold NP-40 lysis buffer (1% vol/vol NP-40, 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with Halt™ Protease and Phosphatase inhibitor Cocktail. The same amount of protein was immunoprecipitated with anti-NEMO and ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology). The IKKα/β kinase activity in the IP complex was tested by using IKKβ Kinase Enzyme Kit (Promega) and following the manufacturer’s instructions.
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