These proteins belong to different families and have different but well-established roles, yet all converge in a common role: involvement in the response to stress. Individually, SOD2 is well known as a major player in the elimination of ROS in all cells while GAPDH has been recognized as promoting resistance to oxidative stress in fungi. The two ion www.selleckchem.com/products/Nilotinib.html transporters identified in this work are important in overcoming the metal ion limitations imposed on invading pathogens by the human or animal host as a defence mechanism and provide the

necessary metal co-factors for SODs and other important proteins. The association of G protein alpha subunits to transport molecules reinforces the role of G proteins in the response to environmental signals and also highlights the involvement of fungal G protein alpha subunits in nutrient sensing in S. schenckii. These interactions suggest

that these permeases could function as transceptors for G proteins in fungi. Methods Strains and culture conditions S. schenckii (ATCC 58251) was used for all experiments. The yeast form of the fungus was obtained from conidia as previously described [76]. S. cerevisiae strains AH109 and Y187 were used for the yeast two-hybrid screening and were supplied with the MATCHMAKER Two-Hybrid System (Clontech Laboratories Inc., Palo Alto, CA, USA). Nucleic acids isolation Total RNA was obtained from S. schenckii yeast cells as described previously by us [25]. Poly A+ RNA was obtained from total RNA using the mRNA Purification Emricasan order Kit from Amersham Biosciences (Piscataway, NJ, USA). Yeast two-hybrid assay MATCHMAKER Two-Hybrid

System was used for the yeast two-hybrid assay using all 3 different reporter genes for the confirmation of truly interacting proteins (Clontech Laboratories Inc.). For the construction of the SSG-1 bait plasmid, a pCR®2.1-TOPO® LY3023414 supplier plasmid (Invitrogen Corp. Carlsbad, CA, USA) containing the ssg-1 gene cDNA sequence of S. schenckii from the laboratory collection was used as template for PCR to obtain the coding sequence of the ssg-1 gene. E. coli TOP10F’ One Shot® chemically competent cells (Invitrogen Corp.) containing the plasmid were grown in 3 ml of LB broth with kanamycin (50 μg/ml) at 37°C for 12 to 16 hours Glycogen branching enzyme and the plasmid isolated with the Fast Plasmid™ Mini kit (Brinkmann Instruments, Inc. Westbury, NY, USA). The ssg-1 insert was amplified by PCR using primers containing the gene sequence and an additional sequence containing an added restriction enzyme site. The Ready-to-Go™ Beads (Amersham Biosciences, GE Healthcare, Piscataway, NJ, USA) were used for PCR. The forward PCR primer included the adapter sequence added at the 5′ end containing the restriction site for Nde I was used to amplify the ssg-1 cDNA. The primers used were: SSG-1/NdeI/(fw) 5′ ccatatggccatgggttgcggaatgagtgtggaggag 3′ and SSG-1 (rev) 5′ gataagaccacatagacgcaagt 3′.