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Roteins in Saccharomyces cerevisiae . Procedures Enzymol. 2002; 344:61731. [PubMed: 11771415] 48. Sprague FG Jr. Assay of yeast mating reaction. Procedures Enzymol. 1991; 194:773. [PubMed: 2005823] 49. Hao N, Nayak S, Behar M, Shanks RH, JAK2 Inhibitor custom synthesis Nagiec MJ, Errede B, Hasty J, Elston TC, Dohlman HG. Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5. Mol. Cell. 2008; 30:64956. [PubMed: 18538663] 50. Ballester R, Marchuk D, Boguski M, Saulino A, Letcher R, Wigler M, Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990; 63:85159. [PubMed: 2121371] 51. Sikorski RS, Hieter P. A method of shuttle vectors and yeast host strains designed for effective manipulation of DNA in Saccharomyces cerevisiae . Genetics. 1989; 122:197. [PubMed: 2659436] 52. Hoffman GA, Garrison TR, Dohlman HG. Endoproteolytic processing of Sst2, a multidomain regulator of G protein signaling in yeast. J. Biol. Chem. 2000; 275:375227541. 53. Elbing K, McCartney RR, Schmidt MC. Purification and characterization on the 3 Snf1activating kinases of Saccharomyces cerevisiae . Biochem. J. 2006; 393:79705. [PubMed: 16201971]NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; offered in PMC 2014 July 23.Clement et al.PageNIH-PA Author ManuscriptFig. 1. Gpa1 is phosphorylated in cells cultured beneath circumstances of low glucose availability(A) Wild-type (WT), reg1, elm1, and diploid yeast strains expressing endogenous GPA1 were grown in yeast extract, peptone, and dextrose (YPD) containing 2 [high (H)] or 0.05 [low (L)] glucose and were analyzed by Estrogen receptor Inhibitor Gene ID Western blotting with an anti-Gpa1 antibody. Therapy with 0.05 glucose was performed for 5 min soon after cells had undergone log-phase development in YPD containing two glucose. Diploid cells don’t have Gpa1 and thus had been used as a adverse handle for the antibody. Gpa1 was detected in two bands indicated by the arrows; the upper band corresponds towards the phosphorylated protein. The asterisk denotes a nonspecific band. (B) Time-course evaluation of Gpa1 phosphorylation. WT, reg1, and elm1sak1tos3 strains have been grown in two glucose (H), have been washed in 0.05 glucose (W), or were grown in 0.05 glucose for the indicated instances (in minutes). Cell lysates have been analyzed by Western blotting with an anti-Gpa1 antibody. (C) Analysis of Gpa1 phosphorylation in yeast strains singly deficient in kinases that phosphorylate Snf1. WT cells and the indicated strains have been treated as described in (A) and were analyzed by Western blotting with anti-Gpa1 antibody. (D) Left: Analysis of Gpa1 phosphorylation in WT cells and within the indicated double and triple kinase eficient strains treated as described in (A). Correct: Impact of reconstitution in the triple kinase eficient strain with plasmid encoding Sak1. Yeast cells deficient in Elm1, Sak1, and Tos3 had been transformed with empty vector (EV) or with plasmid encoding Sak1, treated as described in (A), then analyzed by Western blotting with antibody against Gpa1. (E) Comparison with the responses with the snf1 strain to higher and low glucose with these of WT cells plus the elm1sak1tos3 strain. Cells were treated and analyzed as described in (A). (F) Impact from the loss of Gpa1 signaling components on its phosphorylation. Leading: WT cells as well as the ste2, ste4, sst2, and vps15 strains had been treated and analyzed as described in (A). Bottom: Shorter exposure with the Western blot shown above. (G) Quantitation of.