The abundances of phosphorylated Gpa1 (pGpa1) protein within the indicated strains exposed to high- or low-glucose circumstances as determined by densitometric evaluation of bands from 3 individual experiments. The quantity of pGpa1 protein in each strain is expressed as a percentage with the volume of total Gpa1 protein. Western blotting information in (A) to (F) are representative of 3 independent experiments, except for (B), which is representative of two independent experiments.NIH-PA CB1 Agonist MedChemExpress Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; accessible in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 2. The protein kinase Sak1 along with the phosphatase regulatory subunit Reg1 act on Gpa(A) Coimmunoprecipitation of Gpa1 and Sak1. WT cells had been transformed with plasmids encoding the indicated proteins and had been cultured below high-glucose (H) or low-glucose (L) circumstances. Cells had been subjected to immunoprecipitation (IP) with an anti-FLAG antibody (FLAG), eluted in SDS-PAGE sample buffer, and then analyzed by Western blotting (IB) to detect coimmunoprecipitated Sak1-TAP with antibody against protein A (Protein A). Cell lysates (input) had been also analyzed by Western blotting together with the indicated antibodies. (B) In vitro kinase assays. Purified TAP fusion proteins of WT Sak1 (Sak1-TAP) or even a kinase-deficient mutant Sak1 (Sak1D277A-TAP) were incubated with or without having purified recombinant Gpa1 protein within the presence of [-32P]ATP. The Sak1-TAP fusion proteins had been purified from a sak1snf1 strain to avoid prospective copurification of Snf1. Left: Autoradiogram showing the incorporation of radioactive phosphate into the indicated proteins. Suitable: The Sak1-TAP input was detected by Western blotting evaluation with antibody against protein A, whereas the Gpa1 input was detected by Coomassie gel staining. (C) Coimmunoprecipitation of Reg1 and Gpa1. WT cells had been transformed with plasmids encoding the indicated constructs and have been cultured beneath high- or low-glucose circumstances. Cell lysates have been subjected to immunoprecipitation with anti-FLAG antibody, eluted in SDS-PAGE sample buffer, then analyzed by Western blotting with an antihemagglutinin (HA) antibody to detect coimmunoprecipitated Reg1-HA. Cell lysates (input) had been also analyzed by Western blotting with all the indicated antibodies. (D) Purified recombinant 6 is-Gpa1 and Reg1-MBP (maltose-binding protein) proteins were combined in vitro and resolved by steric exclusion chromatography. Proteins had been detected by Western blotting analysis with antibodies particular for Gpa1 or MBP. All information are representative of two independent experiments.Sci Signal. Author manuscript; accessible in PMC 2014 July 23.Clement et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. three. Snf1-activating kinases limit early mating responses, whereas Reg1 promotes maximal mating responsesNIH-PA Author Manuscript(A) Early og phase cultures of WT and Bcl-xL Inhibitor custom synthesis elm1sak1tos3 cells were left untreated or treated with 3 -factor (-F) for the indicated instances just before samples had been harvested. Best: Western blotting evaluation of samples with antibody against phosphorylated p44/42 MAPK (to detect p-Fus3 and p-Kss1), antibody against total Fus3 protein, and antibody against Gpa1. Glucose-6-phosphate dehydrogenase (G6PDH) was applied as a loading control. Bottom: Densitometric evaluation of the abundance of p-Fus3 in each sample normalized to the abundance of total Fus3 protein. Data.
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