, as well as the sequences had been identified by browsing the SwissProt database.the Bradford method (BioRad), and also the samples had been analyzed by SDS-PAGE.Cell Wall Protein ExtractionThis procedure was performed as described in da Silva Castro et al., [57], with some modifications. Yeast cells had been frozen in liquid nitrogen and disrupted by maceration, plus the material was lyophilized, weighed and resuspended in 50 mM Tris buffer. The supernatant was separated from the cell wall fraction by centrifugation at 10000 6 g for ten min at 4uC. To take away noncovalently linked proteins and intracellular contaminants, the isolated cell wall fraction was washed extensively with 1 M NaCl and boiled 3 occasions in SDS extraction buffer (50 mM Tris?HCl, pH 7.8, 2 w/v SDS, 100 mM Na DTA, and 40 mM bmercaptoethanol) and pelleted just after the extractions by centrifugation at 10000 six g for ten min [67]. The protein concentration of the extract was quantified by the Bradford process (BioRad), and the samples have been analyzed by SDS-PAGE.Western Blot AnalysisThe cell wall protein extracts and purified 14-3-3 recombinant protein separated by one- and two-dimensional electrophoresis had been transferred to nitrocellulose membranes.774212-81-6 web The membranes have been incubated with all the polyclonal antibody obtained against the 14-3-3 recombinant protein and peroxidase-conjugated anti-rabbit IgG because the secondary antibody. The reaction was developed having a chromogen substrate consisting of 0.005 g of diaminobenzidine (DAB) diluted in 30 mL of PBS plus 150 mL of hydrogen peroxide. The unfavorable handle reaction was performed with non-immune rabbit serum.Mice and IT InfectionC57BL/6 mice have been obtained in the Isogenic Breeding Unit (Departmento de Imunologia, Instituto de Ciencias Biomedicas, ^ ?Universidade de Sao Paulo, Sao Paulo, Brazil) and utilised at 8 to 12 weeks of age.DBCO-amine structure The mice were anesthetized and subjected to intratracheal (IT) P.PMID:23991096 brasiliensis infection as previously described [68]. Briefly, after intraperitoneal anesthesia, the animals were IT infected with 106 P. brasiliensis yeast cells in 50 mL of PBS. At 72 h and four weeks postinfection, the lungs were removed and fixed to analyze the subcellular localization of P. brasiliensis 14-3-3 protein. These experiments had been approved by the Ethics Committee on Animal Experiments of your University of Sao Paulo, Sao Paulo, Brazil.Subcellular Localization with the 14-3-3 Recombinant Protein in P. brasiliensis Yeast Cells in vitro and in vivoTo identify the subcellular localization on the 14-3-3 protein of P. brasiliensis, we performed immunocytochemistry in the ultrastructural level using immunogold labeling. For each and every experiment, both pneumocytes infected with P. brasiliensis (108 cells/mL) for two, 5 and 8 h and lungs removed from C57BL/6 mice IT infected with P. brasiliensis (106 cells/mL) were fixed (two.5 v/v glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) for 24 h at 4uC and submitted for the electron microscopy service of the Institute of Biomedical Sciences (ICB-I) USP-SP for the preparation of ultrathin sections. Following fixation, the cells had been rinsed numerous times applying precisely the same buffer, and cost-free aldehyde groups have been quenched with 50 mM ammonium chloride for 1 h, followed by block staining within a option containing 2 (w/v) uranyl acetate in 15 (v/v) acetone for 2 h at 4uC (four). The material was dehydrated in a series of increasing concentrations of acetone (30 to one hundred v/v) and embedded in LR Gold resinAntibody ProductionPurified reco.