3% w/v proteose peptone, 0.5% w/v beef
extract, 0.5% w/v NaCl, 4% w/v glucose, 1% w/v agar pH 7.2). Preparation of protein extracts from Paracoccidioides spp Total protein extracts from Paracoccidioides spp mycelium and yeast cells were prepared as previously described . Mycelium and yeast cells were frozen and ground with a mortar and pestle in buffer (20 mM Tris–HCl pH 8.8, 2 mM CaCl2) with protease inhibitors (50 μg/mLN-α-ρ-tosyl-L-lysine chloromethylketone; 1 mM 4-chloromercuribenzoic acid; 20 mM leupeptin; 20 mM phenylmethylsulfonyl fluoride; and 5 mM iodoacetamide). The mixture was centrifuged at 10,000 × g at 4°C, for 20 min, and the supernatant was collected and stored at −20 °C. Yeast-secreted proteins of Paracoccidioides spp selleck inhibitor were prepared. Culture supernatant of yeast cells was obtained after 24 h incubation in liquid Fava Netto’s medium. The cells were separated by centrifugation at 5,000 × g for 15 min, and the supernatant was filtered in 0.45 and 0.22 μm filters (MilliPore). Each 50 mL of culture supernatant was concentrated to 500 μL in 25 mM Tris–HCl pH 7.0, and a protease inhibitor was added. The protein concentration of all of the samples was determined according to Bradford . Preparation of protein extracts from macrophage J774 A.1 mouse macrophage
cells purchased from a Cell Bank in Rio de Janeiro, Brazil , were cultured in RPMI 1640 supplemented with fetal bovine serum, nonessential amino acids and interferon gamma (1 U/mL). To obtain the protein extract, cells were detached with 0.9% saline solution find more Sclareol containing trypsin and were centrifuged at 5,000 × g for 10 min. Then, milliQ water was added to lyse the cells, and the solution was centrifuged again. Buffer (20 mM Tris–HCl pH 8.8, 2 mM CaCl2) and protease inhibitors were added to the pellet. Protein concentration was determined according
to Bradford . Heterologous expression and purification of recombinant PbMLS PbMLS recombinant protein was obtained as described by Zambuzzi-Carvalho et al. and Neto et al. . PbMLS cDNA was cloned into the expression vector pGEX-4-T3 (GE Healthcare®, Chalfont St Giles, UK). E. coli (BL21 Star™ (DE3) pLys, Invitrogen, Grand Island, NY) was transformed with pGEX-PbMLS construction by thermal shock and was grown in LB medium supplemented with ampicillin (100 μg/mL) at 20°C until reaching the optical density of 0.6 at 600 nm. Synthesis of the recombinant protein was then initiated by adding isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO) to a final concentration of 0.1 mM to the growing culture. After induction, the cells were PI3K inhibitor incubated for 16 h at 15°C with shaking at 200 rpm. Cells were harvested by centrifugation at 10,000 × g for 10 min. The supernatant was discarded, and the cells were resuspended in 1× phosphate-buffered saline (PBS) (0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4). E.