Correct insertion of hph-un-24 constructs were confirmed by yeast genomic DNA extraction [61] and PCR amplification with primers that flank GAL1. The PA(FLAG) construct was made by fusing a standard FLAG epitope in-frame between hph and the un-24 PA incompatibility domain. The control(FLAG) construct was made by in-frame fusion of the FLAG epitope to the 3′ end of hph. Strains that carried these click here FLAG constructs in a SSA1 knockout background were obtained by mating YAL005CΔ (Additional file 2: Table
S3) separately to yeast strains containing PA(FLAG) and control(FLAG) constructs, random sporulation [59], and selection of double mutants on 200 μg/mL G-418 (Bioshop, Oakville, ON) and hygromycin B. Microscopy, Growth Rate and Minimum Inhibitory Concentration (MIC) Cells were examined by phase-contrast with a Zeiss Axiovision II microscope (Toronto, ON). Use of neutral red as a pH-sensitive stain was previously described [22]. The frequency of cells that had a red-stained cytoplasm (as opposed to selleck products those with a bright red central vacuole only) was determined using a double-blind approach. Cell size was determined as previously described [62] based on cell measurements taken from micrographs of randomly selected
fields of view. The number of cells in 1 mm diameter colonies of similar height was determined by resuspending the colony in 0.1 M NaCl and cell counts using a haemocytometer. Minimum inhibitory concentration (MIC) values for hygromycin B and hydroxyurea (Bioshop, Lot#1932H) were determined using standard methods as previously described [63]. The MIC was recorded as the lowest concentration of inhibitor mafosfamide at which no growth was visible after 2 days incubation at 30°C. Detection of FLAG-tagged Selleckchem GSK2879552 Proteins and Rnr1p Mid-log phase cells grown in YPRaf/Gal were harvested, washed once with ddH2O, and resuspended in
either a) non-reducing extraction buffer [20 mM Tris HCl (pH 7.9), 10 mM MgCl2, 1 mM EDTA, 5% glycerol, 0.3 M ammonium sulphate, 1 mM PMSF and 1 Complete Mini-Protean tablet (Roche, Mississauga, ON)], or b) reducing buffer [20 mM Tris HCl (pH 7.9), 10 mM MgCl2,1 mM EDTA, 5% glycerol, 0.3 M ammonium sulphate, 10 mM DTT, 1 mM PMSF and 1 Complete Mini-Protean tablet]. Cells were lysed using 0.5 mm silica beads and 30 seconds of vigorous vortexing followed by cooling on ice for 2 minutes. This bead vortexing was repeated four times. Cell debris was removed through centrifugation at 16,000 × g for 1 hour at 4°C. Proteins were quantified using a Bradford assay. Cytosolic protein was combined with 2X Laemmli buffer (125 mM Tris HCl (pH 6.8), 20% glycerol, 4% SDS, 0.004% bromophenol blue, with or without 15.4 μg/mL DTT and 0.