Dmem high glucose medium

Cell culture and stable isotope labeling HEK293 cells (human embryonic kidney cells) were incubated under standard culture conditions at 37 °C in a humidified atmosphere enriched with 5% CO 2 in DMEM high-glucose medium (Capricorn Scientific) with 10% dialyzed FBS. For SILAC, cells were cultured in DMEM for SILAC (Thermo Fisher) containing 10% dialyzed FBS, devoid of Arg and Lys and supplemented with either 84 mg l -1 Arg-10 and 146 mg l -1 Lys-8 (H SILAC medium) or the same concentrations of Arg-6 and Lys-4 (M SILAC medium) (Cambridge Isotope Laboratories). Before the experiment, cells were treated with H SILAC medium for 10 days. To monitor protein turnover in mitoribosome assembly intermediates, cells were briefly rinsed with PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 and 1.8 mM KH 2 PO 4 ) and then chased on M SILAC medium for 1.5, 3, 6 or 12 h or used without a chase (0 h). To assess global protein turnover in the total cell or mitochondrial fraction, cells were first chased for 12 h in M SILAC and collected immediately (0 h) or additionally chased in standard DMEM containing L isotopes for 3, 6, 9, 12, 15, 18, 21 and 24 h. After harvesting, the cell pellets were stored at -80 °C until further processing.