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RAW264.7 macrophages (2 × 106) were seeded in 60 mm cell culture plates. After overnight incubation cells were initially treated with IKKβ inhibitor (25 μM, SC-514, Sigma) for 1 h to inhibit NF-kB signaling pathway and then cells were treated with LPS (1 μg/mL) and incubated for additional period of time 4 h70. Cells were harvested, total RNA was isolated using TRIzol reagent, reverse transcribed to cDNA and analyzed by qPCR. Protein was also extracted (SC-514 1 h and additional 1 h LPS treatment) after cell harvesting for Western blot30.
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