Back to article: Stable and destabilized GFP reporters to monitor calcineurin activity in Saccharomyces cerevisiae

FIGURE 1: GFP and GFPPEST function as reporters of calcineurin activity.(A) Schematics of pAMS366-4XCDRE-lacZ, pAMS366-4xCDRE-GFP and pAMS366-4xCDRE-GFPPEST plasmids encoding reporters for CN activity. (B) CN activity was determined via β-gal activity or via flow cytometric quantification of GFP fluorescence intensities in exponentially growing wild type and Δcrz1 cells equipped with either pAMS366-4xCDRE-lacZ, pAMS366-4xCDRE-GFP or pAMS366-4xCDRE-GFPPEST. Values are shown as fold of Δcrz1 cells. Means ± SEM, n = 4. (C) CN activity was measured as in (B) in exponentially growing wild type, Δpmr1, Δcnb1 and Δcrz1 cells equipped with the indicated reporter plasmid. For stimulation of CN activity, cells were treated with 50 mM Ca2+ 1 h prior to measurement. Fold of untreated wild type cells is shown. Dead cells were excluded from the analysis via propidium iodide (PI) staining. Means ± SEM, n = 4. (D) Representative immunoblots of protein extracts from cells described in (C). Immunoblots were decorated with antibodies against β-gal or GFP, respectively, and Pgk1 as loading control. (E-G) Histograms of cells quantified in (C) indicating the shift in green fluorescence intensity of wild type cells with and without 50 mM Ca2+ treatment and Δpmr1 cells equipped with the GFP (E) or the GFPPEST reporter (F, G).

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