L lysine 13c615n2hydrochloride

HCT116 (Ki-67-APEX2) and HCT116 (unmodified) were cultured in SILAC medium (McCoy’s 5A Media for SILAC, Thermo Fisher Scientific): light (L-lysine monohydrochloride (Sigma-Aldrich) l-arginine monohydrochloride (Sigma-Aldrich) (Ki-67-APEX2) and heavy (L-lysine-13C6,15N2 hydrochloride (Sigma-Aldrich) (HCT116, unmodified) respectively for at least 6 passages respectively for at least 6 passages. The cells were treated with 2 mM thymidine for 18 h, and 2.5 mM biotin was added for 30 min; then, the H₂O₂ was added for 1 min, and the reaction was stopped by the addition of stop buffer. Cells were then counted and mixed 1:1 (control; APEX). Cells were lysed for 30 min on ice, sonicated and then incubated for 1 h at 4 °C with streptavidin beads (Pierce). The beads were then washed 3 times with lysis buffer (50 mM Tris-HCl, 0.5% IGEPAI, 200 mM NaCl), and the biotinylated proteins were eluted with sample buffer and processed for mass spectrometry analysis.

For SILAC experiments, epidermal stem cells were cultured for 6 days in R/K-deficient SILAC FAD medium (DMEM:F12 for SILAC, Thermo Scientific #88215 mixed 1:1 with R/K-deficient DMEM, Gibco A14431-01) supplemented with 10% dialyzed serum (Sigma-Aldrich, F0392) and 800 mM L-Lysine ¹³C₆¹⁵N₂Hydrochloride and 482 mM L-Arginine ¹³C₆¹⁵N₄ hydrochloride (Sigma-Aldrich) for “heavy”-labeled media or 800 mM L-Lysine ¹²C₆¹⁴N₂-Hydrochloride and 482 mM L-Arginine ¹²C₆¹⁴N₄ hydrochloride for “light”-labeled media^{27 (link)}.

HEK293T and HeLa cells were maintained in 1× MEM + Glutamax media supplemented with 10% FBS, 1× NEAA, 1× sodium pyruvate, and 1× Pen-Strep. U2OS and RAW 264.7 cells were cultured in 1× DMEM media supplemented with 10% FBS and 1× Pen-Stre. (Gibco catalog number as in Materials and Methods).
For Localis-rex protocol, SILAC HEK293T cells were cultured and passaged at least five times (more than 2 wk) before using. The culturing media consisted of, in final concentrations, SILAC drop-off media 1× DMEM (Thermo Fisher 89985), 10% dialyzed FBS (Sigma-Aldrich F0392), 1× sodium pyruvate (Gibco 11360070), 1× Pen-Strep (Gibco 15140122), and the corresponding light/heavy amino acids. For light amino acids, in final concentrations, 146 μg/mL of L-lysine (Sigma-Aldrich L8662) and 84 μg/mL of L-arginine (Sigma-Aldrich A8094) were used and the same concentrations for heavy amino acids, L-lysine-¹³C₆, ¹⁵N₂ hydrochloride (Sigma-Aldrich 608041) and L-arginine-¹³C₆, ¹⁵N₄ hydrochloride (Sigma-Aldrich 608033), respectively. Other culturing/handling methods are the same as for normal HEK293T cells.