A3361

10 μg protein of the microsomal fractions was separated on precast NuPAGE 4–12% BisTris gels using MOPS running buffer (Life Sciences), followed by transfer onto polyvinylidene fluoride membranes (Millipore) according to the manufacturer's instructions. After blocking in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) supplemented with 5% nonfat dry milk and 0.1% Tween-20 (Sigma), blots were incubated for 1 h with primary polyclonal anti-ATP13A2 (1/1,000 dilution; A3361, Sigma) and anti-GAPDH antibodies (1/5,000 dilution; G8795, Sigma) and 45 min with horseradish peroxidase conjugated IgG secondary antibodies (1/2,000 dilution; Bioke). Expression was detected using enhanced chemiluminescence substrate (Pierce) and the Bio-Rad ChemiDoc MP imaging system. Quantification was performed with ImageJ software (http://rsbweb.nih.gov/ij/).

15 cm ø Petri dishes of transiently transfected COS-8 cells were harvested 48 h post transfection. Subcellular fractions were obtained after differential centrifugation as previously described [37 (link)]: the nuclear fraction (1,000 g, 10 min, 4°C), the mitochondrial/lysosomal fraction (12,000 g, 20 min, 4°C) and the microsomal fraction (200,000 g, 35 min, 4°C). To prepare total membrane fractions, the nuclear fraction was removed (1,000 g, 10 min, 4°C) and the remaining lysate was centrifuged for 35 min at 200,000 g (4°C). Pellets were suspended in 0.25 M sucrose supplemented with protease inhibitors (SigmaFast, Sigma). Protein concentrations were determined via the Qubit fluorometric method (LifeTechnologies).
Immunoblotting was performed as previously described [37 (link)]. Membranes were probed with primary rabbit polyclonal antibodies directed against ATP13A1 (SY2559, homemade) or ATP13A2 (SY3072, homemade [37 (link)] or A3361, Sigma) or with a mouse monoclonal antibody directed against mCherry (AB125096, Abcam) or GAPDH (G8795, Sigma). Proteins were detected using enhanced chemiluminescence (Supersignal^TM West Pico, LifeTechnologies) and the ChemiDoc^TM MP Imager (Bio-Rad). For detection of Ypk9p a monoclonal anti-FLAG antibody was used (F3165, Sigma) which was recognized by an anti-mouse antibody coupled to alkaline phosphatase (A4312, Sigma).