After stimulation, cells were lysed in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM sodium fluoride, and 25 mM sodium pyrophosphate] supplemented with phosphatase inhibitors (Roche). The protein lysates were mixed with NuPAGE LDS sample buffer (Novex), resolved by SDS–polyacrylamide gel electrophoresis, and analyzed by Western immunoblotting with anti–phospho-TBK1 antibody (D52C2Cell Signaling Technology) and anti-TBK1 antibody (D1B4, Cell Signaling Technology).

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