Protein identification based on MS MS spectra was

Protein identification based on MS/MS spectra was performed by searching the Swissprot database with the Mascot ver. 2.3 engine [3]. Searching limiters included propionamide cysteine as a fixed modification as well as methionine oxidation and deamidation of glutamine and asparagine as variable modifications, a precursor ion m/z tolerance of 10ppm and trypsin as proteolytic enzyme, with up to one missed cleavage. Only doubly- and triply-charged precursor ions were considered, using an error of 0.2 Da for matching daughter ions in the MS/MS spectra. The false discovery rate (FDR) was set to 1% for peptide and protein identifications. In the case of proteins identified from a single peptide, the ESI-MS/MS spectra were manually inspected and the identification considered reliable only if four or more consecutive C-terminal yn′′ fragment ions were assigned to intense signals and complementary bn ions were detected. Data of protein identification is presented in Supplementary file S1.
Ninety-six well plates (Costar, 3597) were seeded with 3×104cells/well of the Huh 7.5 line and incubated 18–24h at 37°C, 5% CO2 until 90% confluence. The DENV2 inoculum was added at a multiplicity of infection (m.o.i) of 0.01 and infection was allowed to proceed for 2h. Non-internalized virions were inactivated by a short treatment with Gly pH 3.0, the infection was allowed to proceed for 24h, and viral yield was evaluated in cell supernatants by plaque formation assays in Vero dcb (Fig. 3). For the assays involving a pre-incubation step at 4°C, the monolayers were pre-chilled for 5min on ice, the indicated dilutions of plasma sample alone or mixed with DENV2 in DMEM w/o FBS were added, and the plates were further incubated for 90min at 4°C. Afterwards, the monolayers were washed twice with DMEM 2% FBS (pre-chilled to 4°C). The plates incubated with virus:plasma sample mixtures were then incubated for 24h at 37°C. Post-infection activity was evaluated by infecting the cells as described above and then, after washing twice with DMEM w/o FBS, adding dilutions of the plasma samples in DMEM w/o FBS and incubating the plates for 6h at 37°C, 5% CO2. Next, the monolayers were washed again and DMEM 2% FBS was added. The supernatants were collected 24h post infection for virus titration in a plaque assay using Vero cells.
Toxicity assay Vero cells were seeded in 96-well plates and treated with indicated dilutions of ELUAXC in DMEM, 2% FBS either for 2h or 6h at 37°C, 5% CO2. At the end of the incubation, cell supernatants were removed and 50μL of 2mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Invitrogen, EE.UU) in PBS was added. Next, cells were incubated for 4h at 37°C, 5% CO2 before solubilization of formazan crystals in 100µL of Dimethylsulfoxide. Absorbance was measured at 540nm. For calculation, cell death induced by 0.1% Triton X-100 was defined as 100% (Fig. 4).
Data
Experimental design, materials and methods
Acknowledgments We thank the Centro Trasfusionale of the Hospital of Padua (ULSS 16) for providing buffy coats. This work was supported by the University of Padova (ex 60%, 2009–2010; PRAT 2009 CPDA095787) and the Italian Ministry of University and Research (PRIN 2010NRREPL). The authors declare no conflict of interest.
Experimental design Therefore, we used a ggt1 mutant line that had been previously characterized [3,4], and imposed a UV-B treatment. In this way, we generated four experimental conditions: 1) untreated, wildtype; 2) untreated, ggt1 mutant; 3) UV-B treated, wildtype; and 4) UV-B treated, ggt1 mutant. Finally, we obtained the extracellular washing fluid (ECWF) with the aim to gain the following information: i) the effect of UV-B treatment on each genotype; ii) differential apoplastic protein composition in ggt1 vs . wildtype; iii) possible differences in the behavior of the ggt1 mutant and the wildtype under UV-B.
Materials and methods