Reagents
Cell culture and SILAC labeling
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
Liquid chromatography–tandem mass spectrometry
MS data analysis
Bioinformatic analysis
Statistical analysis
Cells were washed in PBS and lysed for 10 min at 99°C in 6 M guanidine-HCl, 100 mM tris (pH 8.5), 5 mM TCEP, and 10 mM CAA, and whole-cell extracts were sonicated. Cell lysates were digested by Lys-C (Wako) and trypsin (Sigma-Aldrich), and resulting peptides were processed by high-pH fractionation (14 fractions per biological replicate; two replicates in total) as described by Batth et al. (75).