Mccoy s 5a media for silac

HCT116 (Ki-67-AID) 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) (auxin) and heavy (L-lysine-13C6,15N2 hydrochloride (Sigma-Aldrich) (control) respectively for at least 6 passages. The cells were treated with doxycycline (2 μg/ml) and 2 mM thymidine, 24 and 18 h before the addition of auxin 1000 μM, respectively. After the 4 h treatment with auxin, the cells were counted and mixed 1:1 (control/auxin). Nuclear isolation was performed as described in [66 ]. Cell lysate was sent for mass spectrometry analysis.

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.