Characterization of cT1R1-NTD using far-UV circular dichroism spectroscopy.

<p>Protein concentration in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM DTT and 0.1 mM DDM was approximately 0.2 mg/ml. Light path: 0.01 cm.</p

Strategy used for expression of the cat T1R1-NTD in bacteria.

<p>(A) The N-terminal domain (NTD) of cT1R1 was expressed independently from the transmembrane heptahelical domain (HD), minus a short putative signal peptide (S), and a cysteine-rich region (CRR). (B) The pET28-cT1R1-NTD plasmid encodes a fusion protein that contains an N-terminal His-tag that can be cleaved with thrombin, followed by cT1R1-NTD (Leu21-Ser495) and a C-terminal His-tag. (C) Full-length cT1R1 is presented according to its primary amino acid sequence deduced from DNA sequence. The numerical positions of amino acid residues of cT1R1 are indicated.</p

cT1R1-NTD binds L-amino acids and IMP.

<p>Normalized maximal fluorescence intensity of cT1R1-NTD before and after addition of ligands (100 μM final concentration). L-Cys does not affect cT1R1-NTD fluorescence. Fluorescence of cT1R1-NTD alone was defined as 100% in absence of ligand. Excitation and emission wavelength were 295 nm and 340 nm, respectively. cT1R1-NTD concentration was 0.5 μM. Data values are the mean ± SEMs of more than nine independent replicates of at least three independently refolded protein samples. *, Significantly different from cT1R1-NTD before addition of ligands (one-way ANOVA followed by Dunnett’s, p ≤ 0.05; for L-Arg p ≤ 0.08).</p

Dissociation constants (<i>K</i>_d) values of ligands for cT1R1-NTD.

<p>Dissociation constants (<i>K</i>_d) values of ligands for cT1R1-NTD.</p

SDS-PAGE analysis of purified cT1R1-NTD inclusion bodies.

<p>cT1R1-NTD is indicated with an arrow while the star indicates a band corresponding to a N-terminal fragment of cT1R1-NTD. The proteins were separated by 12% SDS-PAGE and stained with Coomassie blue. The molecular mass markers are in lane M.</p

SEC-MALS analysis of cT1R1-NTD.

<p>The column was equilibrated and cT1R1-NTD eluted with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM DDM. The chromatograms show the readings of the light scattering (LS), the differential refractive index (dRI) and UV detectors in red, blue and green, respectively. The scale for the LS detector is shown in the right-hand axis. The thick black line indicates the calculated molecular mass of the eluting protein throughout the chromatogram (scale on the left-hand axis). cT1R1-NTD has a fitted molecular mass of 52.5 kDa; its theoretical monomer molecular mass value is 55.4 kDa.</p

Cell assay detection of plastic-bound prions.

<p>Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrP^res in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrP^res levels in ovRK13 cultures subjected to the standard cell assay (no coating). M are standard molecular mass marker proteins (20, 30 and 40 kDa).</p

Sensitivity of moRK13 cell assay for the detection of RML mouse prions.

<p>Serial 10-fold dilutions (from 10⁻⁴ to 10⁻⁸) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020563#pone-0020563-g001" target="_blank">figure 1D</a>. PrP^res was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.</p

Sensitivity of ovRK13 cell assay for the detection of PG127 ovine prion.

<p>A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. <i>Right panel:</i> Serial 10-fold dilutions (from 10⁻⁴ to 10⁻⁶) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrP^res by immunoblotting. Positive transmission was detected for dilutions up to 10⁻⁵. No PrP^res was observed when inoculated ovRK13 did not express the ovine PrP (dox-). <i>Left panel:</i> total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10⁻⁵ to 10⁻⁷) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrP^res in a 1^st set of inoculated cultures was isolated (1^st round) while cultures of the 2^nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrP^res was isolated (2^nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).</p