Anner (Li-Cor Biosciences). Main antibodies and dilutions applied have been: rabbit anti-HA, 1:1000 (Covance Inc., Dedham, Massachusetts, United states); mouse anti-HA, 1:1000 (Covance Inc.); mouse anti-FLAG, 1:5000 (Sigma ldrich, St. Louis, Missouri, United states of america); rabbit antiFLAG, 1:5000 (Sigma ldrich); tissue culture medium containing mouse anti-c-myc mAb 9E10, 1:one hundred (Monoclonal Antibody Facility, Cancer Investigation Laboratory, University of California, Berkeley); rabbit anti-Ypk1(P-T662), 1:20,000 (generous gift from Ted Powers, University of California, Davis); and, rabbit anti-yeast Pgk1, 1:10,000 (this laboratory).Protein purification and in vitro kinase assayYpk1 and GST-Fps1(531-0669) proteins were purified as previously described (Muir et al., 2014). Following protein purification, Ypk1 in vitro kinase assays were performed as previously described (Muir et al., 2014).Measurement of intracellular glycerol accumulationMeasurement of intracellular glycerol was performed as described (Albertyn et al., 1994a). Briefly, samples (40 ml) of exponentially-growing cultures have been harvested by centrifugation, washed with 1 ml of medium, recollected along with the resulting cell pellets frozen in liquid N2 and stored at -80 priorMuir et al. eLife 2015;4:e09336. DOI: 10.7554/eLife.9 ofResearch advanceBiochemistry | Cell biologyto analysis. Each and every cell pellet was boiled for 10 min in 1 ml of 50 mM Tris-Cl (pH 7.0). This eluate was clarified by 870823-12-4 custom synthesis centrifugation for 15 min at 13,200 rpm (16,100 ) inside a microfuge (Eppendorf 5415D). Glycerol concentration within the resulting supernatant fraction was measured using a commercial enzymic assay kit (Sigma Aldrich) and normalized for the protein concentration with the exact same initial extract as measured by the Bradford approach (Bradford, 1976).Fluorescence microscopy of Fps1-GFPAn fps1 strain was transformed with plasmids expressing wild-type Fps1-GFP or the mutant Fps1-GFP derivatives and grown in selective medium to mid-exponential phase. Samples with the resulting cultures were viewed straight under an epifluorescence microscope (model BH-2; Olympus America, Inc.) working with a 100objective fitted with appropriate band-pass filters (Chroma Technologies Corp.). Images were collected using a CoolSNAP MYO charge-coupled device camera (Photometrics, Tucson, Arizona, United states of america).Co-immunoprecipitation of Fps1 and RgcCo-immunoprecipitation experiments were performed with minor modifications as previously described (Lee et al., 2013). Cells expressing Fps1-3xFLAG (yAM271-A), Fps13A-3xFLAG (yAM272-A) or untagged Fps1 (BY4742) had been transformed with empty vector or exactly the same vector expressing Fps1-3xFLAG (pAX302) or Fps13A-3xFLAG (pAX303) below manage on the MET25 promoter. These transformants have been then cotransformed with a plasmid expressing Rgc2-3xHA below manage on the MET25 promoter (Lee et al., 2013). Cultures of each and every were grown to mid-exponential phase in SCD-Ura-Leu. Cultures were then diluted to A600 nm = 0.two in 1 l of SCD-Ura-Leu-Met to induce 10083-24-6 In Vivo expression of Rgc2-3xHA and Fps1-3xFLAG and grown at 30 for four hr. Cells were harvested by centrifugation and resuspended in 5 ml of TNE+Triton+NP-40 (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, four mM NaVO4, 50 mM NaF, 20 mM Na-PPi, five mM EDTA, five mM EGTA, 0.5 Triton-X100, 1.0 NP-40, 1cOmplete protease inhibitor [Roche, Pleasanton, California, United States]). The cells were then lysed cryogenically utilizing Mixer Mill MM301 (Retsch GmbH, Haan, Germany). The lysate was thawed on ice and after that clarified by.
Posted inUncategorized