Sepharose6 beads

Cells were transfected with Polyfect (Qiagen) and used for experiments after 24 hours. For lysis, cells were resuspended in Nonidet-40 lysis buffer (1% NP-40, 50mM Hepes pH7.4, 250 mM NaCl, 5 mM EDTA, Halt phosphatase inhibitor cocktail (ThermoScientific), one tablet of EDTA-free protease inhibitor (Roche) per 50 ml) on ice for 5 min. Lysates were cleared by centrifugation in a microcentrifuge (13000r.p.m., 10min, 4C). Proteins were quantified with BCA (Pierce). For immunoprecipitations, lysates were precleared on Sepharose6 beads (Sigma) (40 min with rotation, 4 °C) and then incubated either with HA-, V5- or FLAG-coupled beads (3h with rotation, 4 °C) or with primary antibody and protein G-sepharose (GE healthcare) (14h with rotation, 4 °C). Beads were recovered and washed four times with lysis buffer before analysis by SDS-PAGE and immunoblotting. When required, a mouse anti-rabbit IgG (conformation specific) antibody was used for immunoblot and revealed with an anti-mouse HRP-conjugated antibody to avoid detection of immunoglobulin heavy chains. In case of detection of endogenous SLC38A9, samples were treated with PNGase (NEB, 250U for 30 ul, 1h, 37 °C) before SDS-PAGE. Nuclear-cytoplasm cell fractionation was performed as previously described³² .

Cells were washed in PBS and lysed in ice-cold HENG buffer (50 mM HEPES-KOH [pH 7.9], 150 mM NaCl, 20 mM Na₂MoO₄, 2 mM EDTA, 5% glycerol, 0.5% Triton X-100, one tablet of EDTA-free protease inhibitor (Roche, Indianapolis, IN, USA) per 50 ml, 20 mM NaF and 0.4 mM Na₃VO₄) for 10 min on ice. Lysates were cleared by centrifugation (13000 rpm, 10 min, 4°C), quantified with BCA (Pierce), and precleared (30 min, 4°C) on Sepharose6 beads (Sigma-Aldrich). Subsequently, lysates were incubated (3h, 4°C) with monoclonal anti-HA agarose antibody (Sigma-Aldrich). Beads were recovered by centrifugation and washed three times with lysis buffer before analysis by SDS-PAGE and immunoblotting.