Quantitative Proteomic Analysis of SNX21

SILAC reagents were purchased from Thermo Fisher with the exception of FCS and SILAC DMEM from Sigma. RPE1 cells virally expressing selected plasmids were seeded in six-well plates in SILAC labelling medium supplemented with dialysed FCS and cultured over a minimum of six passages to achieve full labelling with respective isotopes and a minimum of two confluent 20 cm dishes for generation of lysates. GFP-expressing control cells were grown in unlabelled medium with standard arginine and lysine (R0K0) and GFP-SNX21-expressing cells were grown in medium supplemented with ‘medium’-mass isotopes [¹³C₆]-arginine and 4,4,5,5-D4-Lysine (R6K4). Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na₃VO₄ and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer (Steinberg et al., 2012 (link); Steinberg et al., 2013 (link); McGough et al., 2014a (link),b (link); McMillan et al., 2016 (link)).