Figure 2. Rac1 forms cellular complexes with the PKA holoenzyme. (A) Schematic view of the principle of the Rluc-PCA based PKA reporter to quantify dynamics of PKA holoenzyme formation. cAMP-elevation triggers RIIβ:PKAc complex dissociation which is accompanied by a decrease of bioluminescence originating from complemented PCA-Rluc fragments (Rluc-F1 and Rluc-F2). (B) Co-immunoprecipiation (IP) of endogenous Rac1 from HEK293 cells stably expressing the Rluc based PKA-PCA sensor followed by bioluminescence analyses (representative of n = 3, ± SEM). (C) GST-PBD pulldown of GTP-Rac1 from HEK293 cells stably expressing the Rluc based PKA-PCA sensor followed by bioluminescence analysis (RLU, relative light units, average of n = 4 independent experiments; ± SEM). Indicated is the percentage of RLU compared with bioluminescence signals obtained in GST-RIIβ pulldowns, performed in parallel.