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. Author manuscript; available in PMC: 2014 Jun 6.
Published in final edited form as: Methods Mol Biol. 2011;756:295–310. doi: 10.1007/978-1-61779-160-4_17

Figure 3. Experimentally verifying the range of targeted CKARs.

Figure 3

A. The range of a kinase activity reporter is determined in three experiments. ‘Basal PKC activity’ is the magnitude of the decrease in FRET ratio upon adding PKC inhibitor after acquiring baseline FRET ratios. ‘PdBu-stimulated activity’ is the FRET ratio increase from baseline upon addition of PdBu. ‘Phosphatase-suppressed PKC activity’ is the FRET ratio increase from the plateau of the maximal response to PdBu upon addition of Calyculin A. FRET ratio changes from GolgiCKAR depicted here are normalized to 1, and represent the average responses of multiple COS7 cells across three or more dishes; maximal FRET ratio changes are determined by fitting the data to monoexponential curves. Experimentally-determined ranges for targeted CKAR responses in COS7 cells. Note that Mito-CKAR has a decreased range compared to all other reporters, which all exhibit an approximately 20% maximal FRET ratio change; this likely results from slight proteolysis of Mito-CKAR in COS7 cells. Adapted from5.