To identify proteins that interact with endogenous ULK1, KT-1 cells were left untreated or were treated with human IFN-γ (5 × 103 IU/ml) for 10 min and then lysed in NP-40 buffer [20 mM Hepes (pH 7.4), 180 mM KCl, 0.2 mM EGTA, and 0.1% NP-40] supplemented with protease and phosphatase inhibitors. Three milligrams of protein (total cell lysates) from untreated and IFN-γ–treated samples was used for IP of endogenous protein-ULK1 complexes using ULK1 (D8H5) rabbit monoclonal antibody–conjugated to magnetic beads (custom order, Cell Signaling Technology). As control, the same procedure was followed for IFN-γ–treated lysates, but using rabbit (DA1E) monoclonal antibody immunoglobulin G (IgG) XP Isotype control–conjugated to magnetic beads (no. 8726, Cell Signaling Technology) instead of the ULK1 antibody. After incubating the samples overnight with rotation at 4°C, the beads were washed two times with NP-40 buffer and one time with washing buffer [20 mM Hepes (pH 7.4), 180 mM KCl, and 0.2 mM EGTA]. Protein-ULK1 complexes were eluted from the beads by incubation with Lane Marker Reducing Sample Buffer (Pierce) at 95°C for 10 min, and proteomic analyses were performed in the Northwestern Proteomics Core Facility (Northwestern University, Chicago). IP-eluted proteins were initially separated using SDS–polyacrylamide gel electrophoresis (PAGE) and cut into 10 equivalent height bands before standard in-gel digestion (66). Resulting peptides were extracted from the gel pieces and desalted using solid-phase extraction on a Pierce C18 Spin column, before elution in 40 μl of 80% acetonitrile in 0.2% formic acid. After lyophilization, peptides were reconstituted with 0.1% formic acid in water and injected onto a trap column {150 μm [inner diameter (ID)] by 3 cm} coupled with a nanobore analytical column [75 μm (ID) by 15 cm; both ReproSil-Pur C18-aQ, 3 μm]. Samples were separated using a linear gradient of solvent A (95% water, 5% acetonitrile, and 0.1% formic acid) and solvent B (5% water, 95% acetonitrile, and 0.1% formic acid) over 60 min. nLC-MS/MS data were obtained on a Velos Orbitrap (Thermo Fisher Scientific) mass spectrometer. Data were searched using Mascot 2.5 (Matrix Science) against the human SwissProt database, and results were reported at 1% false discovery rate in Scaffold 4.5 (Proteome Software). Proteins identified by nLC-MS/MS analysis in the control group (rabbit IgG) were excluded from our data analysis.

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