Assays

For diGly proteomics: HeLa cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine (38 μg/mL) and arginine (66 μg/ml). Medium and heavy media were the same except the light lysine was replaced with K4 and K8-lysine, respectively. Medium and heavy labeled cells were treated for 1 h with 250 µM LLOMe while light labeled were treated for 1 h with vehicle alone (EtOH). Light and heavy ...

HeLa cells stably expressing CNN2-APEX2 were grown at 37°C in DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate. Cells were differentially treated with 5 µM NMS-873 for 15 min followed by 1 h 1 mM LLOMe (Sigma) and 2 h washout wihout any drugs. Proximity labeling was performed essentially as described before (Korver et al., 2019). Briefly, cells were incubated with 500 µM Biotin-Phenol during the last 30 min and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop ...