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. 2015 Jun 23;5:11133. doi: 10.1038/srep11133

Figure 4. Analyses of PKA reporter sensitivity and its miniaturization using human cells.

Figure 4

(a) Time-dependent evaluation of complex formation of RIIb-F[1]:PKAc-F[2] hours post transfection (1 h, 2 h and 4 h). The effect of forskolin (50 μM; 10 min) on PKA complex formation has been determined. (b) Fold increase of luminescence signals originating from the complemented Rluc PCA based PKA reporter following transient overexpression in HEK293 cells for indicated time-frames is shown. Immunoblotting shows expression levels of endogenous and overexpressed PKA subunits. The same membrane has been probed first with RIIb and then with PKAc antibodies (BD Biosciences; #610626, #610981). (c) Dose-dependent effects of forskolin exposure on PPI-mediated luminescence measured simultaneously in 1536-well plate format. HEK293 cells transiently expressing the PKA reporter were treated for 5 min with 50 μM forskolin, transferred to a 1536 well plate and luminescence in the presence of benzyl-coelenterazine (CTZ) was captured for 30 sec on the Fusion imaging platform (Biorad). The pseudo-color scale indicates intensities of emitted luminescence signals. The quantification summarizes the effect of forskolin exposure on RIIb:PKAc interaction.