L lysine

U2OS, HCT116, and HEK293T cells were obtained from ATCC and cultured in D-MEM medium (U2OS and HEK293T) or RPMI 1640 (HCT116) supplemented with 10% fetal bovine serum, l-glutamine, penicillin, and streptomycin. OCI-AML3 cells were purchased from DSMZ GmbH and cultured in a D-MEM medium (PAN-Biotech) containing 20% FBS, l-glutamine, penicillin, and streptomycin. Cells were routinely tested for mycoplasma infection with a PCR-based method. For SILAC labeling, cells were cultured in media containing either l-arginine and l-lysine, l-arginine [13C6], and l-lysine [2H4] or l-arginine [13C615N4] and l-lysine [13C6-15N2] (Cambridge Isotope Laboratories)⁷⁹ . All cells were cultured at 37 °C in a humidified incubator containing 5% CO2.

HEK cells were SILAC-labeled in medium containing l-arginine and l-lysine or l-arginine-U-¹³C₆-¹⁵N₄ and l-lysine-U-¹³C₆-¹⁵N₂ (Cambridge Isotope Laboratories, Tewksbury, MA, USA) as previously described [52 (link)]. Peptides were analyzed on a quadrupole Orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanoflow HPLC system (Thermo Scientific), as previously described [53 (link)]. Peptides were loaded onto C18 reversed phase columns (15 cm length, 75 μm inner diameter) and eluted with a linear gradient from 8–40% acetonitrile containing 0.5% acetic acid. The mass spectrometer was operated in a data-dependent mode, automatically switching between MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300–1 750) were acquired in the Orbitrap. The 10 most intense ions were sequentially isolated and fragmented by higher-energy C-trap dissociation (HCD [54 (link)). An ion selection threshold of 50 000 counts was used. Peptides with unassigned charge states, as well as with charge state less than +2 were excluded from fragmentation. Fragment spectra were acquired in the Orbitrap mass analyzer (Thermo Fisher Scientific).

U2OS, HEK293T, RPE-1, and HaCaT cells were obtained from ATCC or DSMZ and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, l-glutamine, penicillin, and streptomycin. Cells were routinely tested for mycoplasma infection with a PCR-based method. For SILAC labeling, cells were cultured in media containing either l-arginine and l-lysine, l-arginine [13C6] and l-lysine [2H4] or l-arginine [13C615N4] and l-lysine [13C6-15N2] (Cambridge Isotope Laboratories)^{50 (link)}. All cells were cultured at 37 °C in a humidified incubator containing 5% CO₂. Cells were transfected with siRNAs using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. The complete list of siRNA sequences and oligos used in this study can be found in Supplementary Tables 1 and 2, respectively.