Kinase assays have been performed by incubating 0.075 to 0.15 pmol of purified TAP
Kinase assays had been performed by incubating 0.075 to 0.15 pmol of purified TAP kinase (corresponding to a final MEK1 manufacturer concentration of three to six nM) and 12.five pmol of recombinant Gpa1 (0.five final concentration) in 1kinase reaction buffer, as described previously forNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; available in PMC 2014 July 23.Clement et al.PageElm1 (6). Reactions had been stopped by the addition of 6SDS-PAGE loading buffer, and samples have been instantly subjected to 10 SDS-PAGE. Gels had been dehydrated and exposed to autoradiography film (HyBlot CL, Denville Scientific). Steric exclusion chromatography of Gpa1 and Reg1 Purified six is-Gpa1 and Reg1-MBP proteins had been subjected to steric exclusion chromatography with an Akta FPLC method in addition to a Sephacryl 2660 S200 column (GE Healthcare). 1 nanomole of 6 is-Gpa1 and three.25 nmol of Reg1-MBP had been equilibrated in 20 mM tris-HCl (pH 8.0), one hundred mM NaCl, 5 glycerol, 1 mM DTT, two mM MgCl2, and 20 GDP. Proteins have been separated at a price of 0.5 mlmin and had been IL-6 Species collected in 7-ml fractions. A 20- sample from each fraction was resolved by SDS-PAGE and analyzed by Western blotting with anti-Gpa1 or anti-MBP antibodies. Pheromone transcriptional reporter assay and quantitative mating assay Transcriptional reporter assays (47) and mating assays (48) were performed as described previously. For the mating assay, equal amounts of early og phase MATa cells (BY4741) and MAT cells (BY4742, leu2 his3 ura3 lys2 MET ) have been mixed, filtered onto nitrocellulose membranes, and incubated on YPD plates containing two or 0.05 glucose. After four hours of incubation, cells have been resuspended and plated onto SCD or SD (synthetic medium containing dextrose) and only LeuHisUra. Mating efficiency was calculated by dividing the amount of diploid colonies by the total number of cells on an SCD plate. Microscopy A microfluidic device was constructed comparable to one particular previously described (49). Cells had been imaged every single five min for 12 hours. Image acquisition was performed with an Olympus spinning disc confocal microscope, and image processing and evaluation had been performed with ImageJ application. Statistical evaluation To assess statistical significance, we made use of Student’s t test for pairwise comparisons. P 0.05 was thought of statistically important. Error bars represent the means SEM of replicates within person experiments.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSupplementary MaterialRefer to Net version on PubMed Central for supplementary material.AcknowledgmentsWe thank M. Carlson and M. Torres for their assistance and encouragement, M. Schmidt for the Sak1 plasmid used for in vitro kinase assays, M. Lee for his early contributions towards the analysis of Reg1, and H. Lien for performing the mating efficiency assays. Funding: This work was supported by NIH grant GM059167 to H.G.D.Sci Signal. Author manuscript; available in PMC 2014 July 23.Clement et al.PageREFERENCES AND NOTES1. Gutkind JS. Regulation of mitogen-activated protein kinase signaling networks by G proteincoupled receptors. Sci. STKE. 2000; 2000 re1. two. Sherwood NM, Krueckl SL, McRory JE. The origin and function with the pituitary adenylate cyclaseactivating polypeptide (PACAP)glucagon superfamily. Endocr. Rev. 2000; 21:61970. [PubMed: 11133067] 3. Dohlman HG, Thorner JW. Regulation of G protein-initiated signal transduction in yeast: Paradigms and principles. Annu. Rev. Biochem. 2001; 70:70354. [Pub.
