Reagents
Cell viability assay
Cell stimulation, lysis, and Western blotting
Immunoprecipitation
RNAi and transfection
Caspase-3/7 activity measurements
Sample preparation for MS-based interactome analysis
Sample preparation for MS-based phosphotyrosine interactome analysis
Sample preparation for MS-based phosphoproteome analysis
TMT labeling and phosphopeptide enrichment
Sample preparation for MS-based proteome analysis
Liquid chromatography–tandem mass spectrometry
MS data analysis
Bioinformatic analysis
Statistical analysis
The human NB cell lines NB1, SH-SY5Y, CLBGA, and NBL-S and the human lung cancer cell line H3122 (provided by S. Mueller, Evotec, Munich, Germany) were cultured in RPMI 1640 including 2 mM l-glutamine (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco). Cell lines were maintained at 37°C in a humidified atmosphere at 5% CO2. For SILAC-based quantitative MS, NB1 cells were labeled in SILAC RPMI (PAA Laboratories) supplemented with 10% dialyzed fetal bovine serum (Sigma-Aldrich), 2 mM l-glutamine (Gibco), and penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco) for at least 2 weeks to ensure complete incorporation of amino acids. Three cell populations were obtained: one labeled with natural variants of the amino acids (light label; Lys0, Arg0) (Sigma-Aldrich), the second labeled with medium variants of amino acids {L-[2H4]Lys (+4) and L-[13C6]Arg (+6)} (Lys4, Arg6), and the third labeled with heavy variants of the amino acids {L-[13C6,15N2]Lys (+8) and L-[13C6,15N4]Arg (+10)} (Lys8, Arg10). Medium and heavy variants of amino acids were purchased from Cambridge Isotope Laboratories.