Before IFN-γ treatment, MEFs were either cultured in 10% FBS-containing medium (for detection of phosphorylation of ERK5, STAT1, and ERK1/2) or starved overnight (for detection of phosphorylation of JNK, MLK3, and p90RSK1). MEFs were then treated with mouse IFN-γ (5 × 103 IU/ml) for 10 or 30 min, as indicated. After treatment, cell pellets were lysed with lysis buffer [50 mM Hepes (pH 7.3), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA (pH 8.0), 100 μM sodium fluoride, 10 μM sodium pyrophosphate, 0.5% Triton X-100, and 10% glycerol) supplemented with protease and phosphatase inhibitors.

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