Nupage 4 12 precast gels

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)).

GFP–INF2–WT was subcloned into pWPXL [pWPXL was a gift from Didier Trono (École polytechnique fédérale de Lausanne, Addgene plasmid # 12257)] and together with packaging vectors pMDG.2 and psAX2 [pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259) and psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260)] transfected into Lenti-X 293T Cell Line (Clontech). Human podocytes were transduced with GFP or GFP–INF2 lentivirus with 8 μg/ml polybrene overnight, the cells were thermo switched and differentiated for a minimum of 10 days [18 (link)]. 2× 175 tissue culture flasks were lysed and GFP and GFP–INF2 protein and interacting proteins were immunoprecipitated using the GFP-Trap system (Chromotek). 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 [19 (link),20 (link)]

For western blotting, cells were lysed in PBS containing 2% Triton X-100 and Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a BCA assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4–12% precast gels (Invitrogen). Proteins were transferred from gels onto PVDF membrane (Immobilon-FL, pore size 0.45 μm, Millipore, catalogue number IPFL00010) for immunoblotting. Transfer was performed in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol) at 100 V for 70 min. Membranes were blocked in 5% milk in PBS containing 0.1% Tween 20 (PBS-T) prior to incubation with the appropriate primary antibodies and fluorescently labelled secondary antibodies. Detection and quantification were carried out using an Odyssey infrared scanning system (LI-COR Biosciences).

All SILAC reagents were sourced from Thermo Fisher; except for dialysed FBS (Sigma). Lentivirally transduced GFP and GFP-SNX3 RPE1 cells were grown in SILAC DMEM for at least 6 passages to achieve full labelling. GFP-expressing control cells were grown in unlabelled medium containing regular arginine and lysine (R0, K0). GFP-SNX3-expressing cells were grown in medium containing ¹³C₆-arginine and 4,4,5,5-D4-lysine (R6, K4). GFP was precipitated with GFP-trap beads (Chromotek) for 60 min. The beads were pooled, proteins were eluted in sample buffer, separated on Nupage 4–12% precast gels (Invitrogen) and subjected to LC–MS/MS analysis on an Orbitrap Velos (Thermo) mass spectrometer^{40 (link)–43 (link)}.

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)).