Qubit fluorometric method

Cells were seeded in 15 cm dishes at a density of 1.5 × 10⁶. After treatment, cells were harvested and resuspended in hypotonic buffer (10 mM Tris-HCl pH 7.5, 0.5 mM MgCl₂, and SigmaFast protease inhibitor cocktail (Sigma)) and a 10 min incubation period on ice. Cells were homogenized by applying 40 strokes in a Dounce homogenizer and after adding the 1 M buffer solution (0.5 M sucrose, 10 mM Tris-HCl pH 7.3, 40 μM CaCl₂, 0.23 μM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (DTT)) 20 additional strokes were performed. The total cell lysate was subjected to differential centrifugation: the nuclear fraction (1,000 g; 10 min), the mitochondrial/lysosomal fraction (12,000 g; 20 min), and, lastly, the microsomal and cytosolic fractions (200,000 g; 35 min; pellet and supernatant, resp.). Microsomal pellets were resuspended in a 250 mM sucrose solution supplemented with protease inhibitor cocktail. All fractionation steps were carried out at 4°C. After solubilization, samples were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C. The protein concentration was determined by the Qubit fluorometric method (Life Technologies).

B. endophyticus and B. anthracis strains (Table 1) were inoculated in 2 ml nutrient broth, followed by overnight incubation at 37 °C. The cells were harvested by centrifugation at 5000 xg for 10 min. Genomic DNA was extracted from the harvested cells using the DNAeasy Tissue kit (Qiagen, Germany) according to the manufacturer’s instructions. The isolated DNA was then quantified using the Qubit® fluorometric method (Life Technologies, USA) according to the manufacturer’s instructions. The DNA integrity was monitored through electrophoreses using a 0.8% agarose gel pre-stained with ethidium bromide and visualized on a UV transilluminator.

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).