13c6 15n4 l arginine hcl

Cells were grown at 37 °C under a humidified 5% CO₂ atmosphere, in a culture medium consisting of high-glucose DMEM (Caisson Labs) for A431, HEK293T, NIH-3T3 and HeLa cells or RPMI (Caisson Labs) for Ramos, HCC827, and A549 cells. The SW620 and MDA-MB-435S cells were grown in L-15 media in a humidified atmosphere with no CO₂ added. All media was supplemented with 10% FBS (Gemini), penicillin (50 IU/mL), streptomycin (50 μg/mL), and glutamine (2 mM) (Cellgro). For SILAC experiments, the culture medium was replaced with either SILAC DMEM or SILAC RPMI (Thermo) as appropriate, and the medium was supplemented with 10% dialyzed FBS, penicillin, streptomycin, and glutamine. For the isotopically heavy cell samples, 100 μg/mL of both [¹³C₆,¹⁵N₄]L-arginine-HCl and [¹³C₆,¹⁵N₂]L-lysine-HCl (Sigma-Aldrich) was also added to the culture medium. For the isotopically light cell samples, the culture medium was supplemented with 100 μg/mL of both L-arginine-HCl and L-lysine-HCl. Cells were passaged at least six times in isotope-containing medium before being used for ABPP-SILAC experiments.