IP studies were performed in HEK293T cells that were transfected using Lipofectamine 2000 (Life Technologies) with epitope-tagged forms of MyD88. MPP or DMSO was added 7 hours after transfection. Twenty hours after transfection, cells were lyzed for 20 min at 4°C in LB [20 mM Hepes/NaOH (pH 7.5), 1.5 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 10% glycerol) supplemented with complete protease inhibitors (Roche Applied Science). After clearance of lysates by centrifugation (10 min, 20,817g, 4°C), lysates were subjected to IP using FLAG M2 resin (Sigma-Aldrich) for 1 hour at 4°C. IP samples and total cell lysates were analyzed by immunoblotting.

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