SILAC-Based Proteomic Analysis of GPR124

Metabolic labeling of amino acids using SILAC was completed as described previously (Lau, Suh, Golkowski, & Ong, 2014 (link); Ong, 2010 ; Ong & Mann, 2006 (link)) with SILAC DMEM media supplemented with 10% dialyzed FBS (Sigma) and either light (L-lysine and L-arginine [Fisher]) or heavy ([¹³C₆, ¹⁵N₂] L-lysine [Sigma-Isotec, St Louis, MO] and [¹³C₆,¹⁵N₄] L-arginine [Cambridge Isotope Laboratories, Andover, MA]) isotope-enriched amino acids. Cells were split into two groups regarded as “heavy” and “light.” SILAC media was applied to cells for at least 5 cell doublings to ensure complete labeling of the proteome, which was verified by mass spectrometry. Membranes were solubilized as above and immunoprecipitation was performed in preparation of mass spectrometry. Each SILAC labeling experiment consisted of two parts completed in parallel: (a) the forward experiment in which a competing myc peptide (5 μg/mL, Sigma) was applied to the heavy condition and (b) the reverse experiment in which the myc peptide was applied to the light condition. Full competition of the GPR124 complex by the myc peptide was verified by western blot analysis (data not shown).