SILAC-based Proteomic Analysis of VPS35

All SILAC reagents were sourced from Thermo Fisher, except for dialyzed FBS, which was from Sigma. Human RPE1 cells were grown in the SILAC DMEM for at least six passages to achieve full labeling (Steinberg et al., 2012; Steinberg et al., 2013 (link)). GFP–VPS35 and GFP were lentivirally transduced before the labeling. Cells were lysed in precipitation buffer (50 mM Tris-HCl, 0.5% NP40, Roche Protease Inhibitor Cocktail), and GFP was precipitated with GFP-trap beads (Chromotek) for 30 min. Samples were separated on Nupage 4–12% precast gels (Invitrogen) and subjected to LC-MS/MS analysis on an Orbitrap Velos (Thermo) mass spectrometer as described previously (Steinberg et al., 2012 (link); Steinberg et al., 2013 (link)).