For knock-down of IRAK-M by siRNA, Raw 264.7 macrophages were transfected with IRAK-M siRNA, and control siRNA (Applied Biosystems) using siPORT NeoFx transfection agent (Applied Biosystems) according to the manufacturer’s instructions. Transfection efficacy was determined by transfecting the cells with GAPDH siRNA. Forty-eight hours after transfection, macrophages were then exposed to 25 mM alcohol for 24 h followed by stimulation with LPS for 6 h. Total RNA extracts and whole cell lysates were analyzed for IRAK-M by real-time PCR and Western blotting to confirm IRAK-M knock-down and supernatants collected were analyzed for TNF-α by ELISA.
