Figure 3
Competitive nucleotide binding assay using fluorescence polarization. Serial dilutions of cAMP or 8-[Ï•-575]-cAMP in the presence of 1 nM 8-Fluo-cAMP were prepared. 2.5 nM R subunit was added and fluorescence polarization was determined at Ex485 nm/Em535 nm. EC50 values for cAMP and 8-[Ï•-575]-cAMP binding to the R subunit isoforms were deducted from the corresponding titration curves. Each data point represents the mean +/- S.E.M. from at least triplicate measurements.