Dmem deficient in l arginine and l lysine

Cells were cultured for 2 weeks in SILAC medium containing DMEM deficient in L-Arginine and L-Lysine (Thermo Fisher Scientific) and supplemented with 10% dialyzed FBS (Biological Industries), 1% P/S, and L-Arginine-HCl [¹³C₆, ¹⁵N₄] (Arg-10)/L-Lysine-HCl [¹³C₆] (Lys-6), or L-Arginine (Arg-0)/L-Lysine (Lys-0) (Thermo Fisher Scientific). Next, 1 × 10⁸ cells for each population were seeded for growing overnight and then washed twice with PBS before harvest.

Cells were cultured for more than 5 doublings in DMEM deficient in L-Arginine and L-Lysine (Thermo Fisher Scientific) supplemented with 10% dialyzed FBS (Biological Industries), 1% P/S, and L-Arginine-HCl [¹³C₆, ¹⁵N₄] (Arg-10)/L-Lysine-HCl [¹³C₆] (Lys-6), or L-Arginine (Arg-0)/L-Lysine (Lys-0) (Thermo Fisher Scientific). Cells were treated with 5 μM MG132 for 2 h before harvest. The cell pellets were lysed at room temperature in urea lysis buffer containing 8 M urea, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and protease inhibitor (Roche). The lysates were cleared by centrifugation at 14,800 rpm for 10 min and lysates containing 15 mg proteins per sample were mixed. Proteins were reduced with 5 mM DTT at 37°C for 45 min and subsequently alkylated with 10 mM iodoacetamide at room temperature for 30 min in the dark. Lysates were diluted to 4 M urea with 50 mM Tris-HCl (pH 8.0), and proteins were digested with Lys-C (Wako) at 37°C for 4 h. The peptide mixtures were further diluted to 1 M urea and digested with Trypsin (Promega) at 37°C for overnight. The reaction was quenched with formic acid, desalted by Sep-Pak PlusC18 cartridge (Waters), and lyophilized.