To confirm the physical interaction between ULK1 and MLK3 identified by nLC-MS/MS analysis, KT-1 cells were left untreated or were treated with human IFN-γ (5 × 103 IU/ml) for 10 min, and U937 cells were cultured overnight in serum-free RPMI 1640 medium and then were left untreated or were treated with human IFN-γ (5 × 103 IU/ml) for 10 min. After treatment, cell pellets were lysed in NP-40 buffer [20 mM Hepes (pH 7.4), 180 mM KCl, 0.2 mM EGTA, 10% glycerol, and 0.1% NP-40] supplemented with protease and phosphatase inhibitors. For IP of endogenous protein-ULK1 complexes, 200 μg of protein (total cell lysates) from each sample was incubated overnight at 4°C with rotation with ULK1 (D8H5) rabbit monoclonal antibody (1:100; no. 8054, Cell Signaling Technology), followed by incubation for 1 hour at 4°C with rotation with protein G Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences). As control, the same procedure was followed using rabbit (DA1E) monoclonal antibody IgG XP Isotype control (no. 3900, Cell Signaling Technology) instead of ULK1 antibody. After IP, the beads were washed three times with NP-40 buffer without glycerol. Protein-ULK1 complexes were eluted from the beads by incubation with Lane Marker Reducing Sample Buffer (Pierce) at 95°C for 10 min. Eluates were resolved by SDS-PAGE and processed for immunoblotting analyses.

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