coli TOP10 One Shot® chemically competent cells. The correct orientation and frame of the inserted gene sequence was verified by
sequencing. The PU-H71 supplier bait containing plasmid was isolated using Fast Plasmid™Mini technology (Brinkmann Instruments) and used to transform competent S. cerevisiae yeast cells (Y187) with the YEAST- MAKER™ Yeast Transformation System 2 (Clontech Laboratories Inc.). Tests for autonomous gene activation and cell toxicity were carried out as described by the manufacturer. A cDNA library using S. schenckii yeast RNA was constructed as described previously in AH109 cells . Transformants were selected in SD/-Leu plates, harvested and used for mating with the bait containing S. cerevisiae strain Y187. Mating of S. cerevisiae yeast cells strains Y187 (Mat-α) and AH109 (Mat-a) was done according to the manufacturer’s instructions as described previously. Colonies growing in triple dropout medium (TDO) SD/-Ade/-Leu/-Trp were tested for growth
in quadruple dropout medium (QDO) SD/-Ade/-His/-Leu/-Trp. These positive colonies were re-plated in QDO medium to verify that they maintained the correct phenotype. Colony PCR was used to corroborate the presence of both plasmids MM-102 cost in the diploid cells using the T7/3′BD sequencing primer pair for the pGBKT7/SSCMK1 plasmid and the T7/3′AD primer pair for the pGADT7-Rec library plasmid and yeast colony suspension as template. The Ready-to-Go™ Beads (Amersham
Biosciences) were used for PCR. The amplification parameters were those described previously . PCR Selleckchem FG4592 products were analyzed on agarose gels and the DNA recovered using Spin-X Centrifuge Tube Filters as described by the manufacturer (0.22 μm, Corning Costar Corp.). The PCR products were cloned and amplified as described previously . Plasmid preparations were obtained using the Fast Plasmid™ Mini technology (Brinkmann Miconazole Instruments) and the inserts sequenced using commercial sequencing services from SeqWright (Fisher Scientific, Houston, TX, USA) and Retrogen DNA Sequencing (Retrogen Inc., San Diego, CA, USA)). Co-immunoprecipitation (Co-IP) and Western blots Co-immunoprecipitation followed by Western blot was used to confirm the interaction of HSP90 identified in the yeast two-hybrid analysis as interacting with SSCMK1 as described previously . S. cerevisiae diploids obtained in the yeast two-hybrid assay were grown in QDO, harvested by centrifugation and resuspended in 8 ml containing phosphate buffer saline (800 μl) with phosphatase (400 μl), deacetylase (80 μl) and protease inhibitors (50 μl), and PMSF (50 μl). The cells were broken as described previously . The cell extract was centrifuged and the supernatant used for Co-IP using the Immunoprecipitation Starter Pack (GE Healthcare, Bio-Sciences AB, Bjorkgatan, Sweden).