The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe

Figure 2

Time- and dose-dependent response to P-factor with the reporter plasmids. (a) Time-dependent response to P-factor with either pAL7-Umam2-GFP-LPI or pAL7-Usxa2-GFP-DSPBC4.01. The cells were exposed to 1 or 0 μM of P-factor and were taken every hour (0–6 h), two hours (6–12 h) or twelve hours (12–24 h). Filled circle depicts OSP210-2 (sxa2Δ, pAL7-Umam2-GFP-LPI) +P-factor, open circle depicts OSP210-2 −P-factor, filled square depicts OSP210-17 (sxa2Δ, pAL7-Usxa2-GFP-DSPBC4.01) +P-factor and open square depicts OSP210-17 −P-factor. The values were the means of triplicate determinations from a typical experiment. The error bars represent ± standard error. (b) Aliquots of culture medium (2 μL) were mounted on slides and visualized by a fluorescence microscope. After the addition of P-factor, the morphology was gradually changed and the production of GFP increased. Scale bar = 10 μm, (c) dose-dependent response. The cells were exposed to the various concentrations of P-factor and were incubated for 24 hours after the addition of P-factor. Filled circle depicts OSP210-2 (sxa2Δ, pAL7-Umam2-GFP-LPI) and filled square depicts OSP210-17 (sxa2Δ, pAL7-Usxa2-GFP-DSPBC4.01). 𝑌-axis denotes a percentage of the maximum response. The values were the means of triplicate determinations from a typical experiment.