Overview
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Key numbers
The worker extracted the full PDF text with layout preservation twice and compared extraction hashes before commit. The following lines are copied from numeric/table-bearing regions of the PDF and retain the source units and wording where legible:
- range. These parameters correlate with the extra and intracellular hASCT2 provides cancer cells with glutamine, one of the major
- plus, Hybond ECL membranes were from GE Healthcare; L- buffer (20 mM Tris HCl pH 7.4, 300 mM NaCl, 10% glycerol,
- (3 H)Glutamine was from Perkin Elmer; anti-ASCT2 (rabbit) was 6 mM β-mercaptoethanol, 0.03% C12 E8 , 1 mM L-glutamine, and
- Recombinant Production of hASCT2 WT 10% glycerol, 6 mM β-mercaptoethanol, 0.03% C12 E8 , 1 mM
- described (Scalise et al., 2018b). For large scale protein Cholesteryl HemiSuccinate (CHEMS) was prepared in 5%
- at final OD of 1 in 250 mL BMMY medium added with 0.5% cycles of 2 min (no pulse, 40 W) with a Vibracell VCX-130
- with glass beads (0.5 mm). After 5 min, almost 90% of the cell
- and, 10% glycerol and homogenized using a potter homogenizer. wise method in which mixed micelles of detergent, protein and
- WT and Mutants mixture containing 100 µL of 10% egg yolk phospholipids (w/v)
- protein and mutants, about 1.5 g of washed membranes (400 µL of 5% C12 E8 or CHEMS as specified in the figure legends,
- glutamine, 10% glycerol, and 2% C12 E8 (w/w) by rotatory stirring µL. All the operations were performed at room temperature.
- Transport reaction was stopped by adding 100 µM HgCl2 ; were prepared using loading dye with 10% SDS and heated for
- Cell Culture, Methyl-β-Cyclodextrin (wavelength = 578 nm). Internal volume in µL was derived from
- Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Molecular Docking Approach
- and a 5% CO2 atmosphere. Prior of methyl-β-cyclodextrin 4.2 (Forli et al., 2016). At first, a blind docking was made,
- was performed when cells reached 70% confluence. Treatment of the grid box was set to 114 × 114 × 114 Å (x, y,
- with 10 mM methyl-β-cyclodextrin was performed for 60 min and z). The optimized ligand molecule was docked into
- solubilized in 500 µL of 1% TX-100 solution. Cell extracts were priority to the lowest binding-energy conformation. As a further
- treated with 0.75% formaldehyde for 10 min shaking at room Other Methods
- prepared in PBS for 5 min shaking at room temperature. Then, was estimated from Coomassie blue-stained 12% SDS–PAGE
- as described in Materials and Methods excluding data of high CHEMS amount, i.e., 100 and 150 µg/mg phospholipids, because out of the plot range. (C) Samples (a
- volume corresponding to 1% of the total reconstitution mixture) derived from (A) were subjected to SDS-PAGE and western blotting analysis, as described in Materials
- on 8% SDS-PAGE. For Western Blot analysis, hASCT2 was transport activity of hASCT2 measured as sodium-dependent
- His antibody 1:10,000 in 3% BSA for 1 h at room temperature Maximal stimulation was observed at a cholesterol concentration
- or with anti-hASCT2 (1:1,000) incubated overnight in 3% BSA of 75 µg/mg total lipids corresponding to 7.5% cholesterol
- secondary antibody anti-rabbi (1: 5,000) in 1% BSA. The reaction in cells (Litvinov et al., 2018); at higher concentrations, the
- increase in transport activity (Figure 1D), as indicated by course data. Data are means ± SD of three independent experiments. In (B),
- HeLa cells were treated with 10 mM methyl-β-cyclodextrin (MCD) as described
- To prove that indeed cholesterol binds to the protein, a 30%, indicating that tryptophan residues might be critical for
- protein extracted from HeLa cells treated with 0.75% formaldehyde as
- immunoblot analysis of purified hASCT2 treated with 1 or 4% formaldehyde as
Methods (brief)
- Cholesteryl HemiSuccinate; β-MCD, Methyl-beta-CycloDextrin; Cryo-EM, Cryo
- glutamine, methyl-β-cyclodextrin (MβCD) and all the other incubation, the Ni-NTA resin was packed into a column and
- from which 3.5 mL were collected.
- containing membrane and cytosolic fractions was collected. The
- and 50 mM Na-gluconate to 100 µL proteoliposomes, at 25◦ C. stopped adding cold 1.25 M glycine solubilized in PBS. Samples
- according to the inhibitor stop method, the same inhibitor 5 min at 65◦ C. Samples were analyzed by SDS—PAGE followed
- was added at time zero to control samples (blanks) (Palmieri by western blotting as described below.
- were eluted with 1 mL 50 mM NaCl and collected in 3 mL of phosphate method as previously described (Indiveri et al.,
- scintillation mixture, vortexed and counted. The experimental 1994). In brief, different proteoliposome samples were prepared
- values were analyzed by subtracting to each sample the respective including, in the reconstitution mixture, 50 mM dipotassium
- stopping the reaction after 15 min, i.e., within the initial linear buffered without phosphate, 100 µL of sample were used
- measure transport rate by first-order rate equation. FeSO4 ). After incubating samples in the dark for 30 min, the
- Cell Culture, Methyl-β-Cyclodextrin (wavelength = 578 nm). Internal volume in µL was derived from
- and a 5% CO2 atmosphere. Prior of methyl-β-cyclodextrin 4.2 (Forli et al., 2016). At first, a blind docking was made,
- with 10 mM methyl-β-cyclodextrin was performed for 60 min and z). The optimized ligand molecule was docked into
- cells were collected and washed with PBS. Cells pellet were stored gels by using the Chemidoc imaging system equipped with
- absence or the presence of 0.75 mg CHEMS prepared as above (Torchetti et al., 2011). The cross-linked samples were analyzed
- as described in Materials and Methods excluding data of high CHEMS amount, i.e., 100 and 150 µg/mg phospholipids, because out of the plot range. (C) Samples (a
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Update history
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