Proteinase K (Sigma Aldrich) was used as positive control. Azocasein assays with significant differences were determined by statistical analysis by using t test. P values of 0.05 or less were considered A-1155463 concentration statistically significant. Preparation and infection of murine macrophages Bone marrow-derived macrophages were obtained by selleck inhibitor flushing the femurs

of 4-12 weeks old female C57BL/6 mice. The cells were cultured as described [34]. Briefly, the obtained cells were cultured for 8 days. The non-adherent cells were discarded and the adherent cells were washed twice with 10 mL of Hank’s Balanced Salt Solution (HBSS). After cells treatment with 10 ug/mL of dispase (Invitrogen) in HBSS (37°C for 5 min), macrophages were removed using a cell scraper and washed in HBSS. Cells were resuspended in RPMI 1640 (106 cells/mL). For infection experiments, 107 P. brasiliensis

yeast cells were added to 2 mL of macrophage suspension and co-cultivated for 24 h (37°C in 6% CO2). The wells were washed twice with HBSS to remove unattached yeast forms. RNA from infected murine macrophages was extracted by using Trizol reagent. ICG-001 in vitro RNAs from uninfected macrophages and from P. brasiliensis yeast cells cultured in RPMI 1640 medium were obtained as control. Quantitative real-time PCR RNA samples were reverse transcribed by using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). The cDNA samples were diluted 1:2 in water, and qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) in the Applied Biosystems Step One Plus PCR

System (Applied Biosystems Inc.). qRT-PCR was performed in triplicate for each cDNA sample. The specificity of each primer pair for the target cDNA was confirmed by the visualization of a single PCR product in agarose gel electrophoresis. The primers and sequences were used as Fossariinae follows: serine-sense, 5′-GGCCTCTCCACACGTTGCTG-3′; serine-antisense 5′-GTTCCAGATAAGAACGTTAGC-3′ and α-tubulin primers: tubulin-sense, 5′-ACAGTGCTTGGGAACTATACC-3′; tubulin-antisense, 5′-GGACATATTTGCCACTGCCA-3′. The annealing temperature for serine and tubulin primers was 60°C. The standard curves were generated by using the cDNAs serially diluted 1:5 from the original dilution. The relative expression levels of genes of interest were calculated using the standard curve method for relative quantification [35]. Statistical analysis was calculated by using t test. P values of 0.05 or less were considered statistically significant. Interaction of PbSP with P. brasiliensis proteins as determined by Two-Hybrid assay Oligonucleotides were designed to clone the complete cDNA encoding the PbSP in the pGBK-T7 (Clontech Laboratories, Inc) expression vector. The nucleotide sequence of the sense and antisense primers were 5′-CATATGATGAAAGGCCTCTTCGCCT-3′ and 5′-CTGCAGTTAAGAGATGAAAGCGTTCTTG-3′, contained engineered NdeI and PstI restriction sites, respectively (underlined).