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. 2018 Jun 4;217(6):2167–2184. doi: 10.1083/jcb.201708053

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© 2018 Isensee et al.

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Figure 3. — High doses of unconjugated Rp-cAMPS isomers are required to inhibit dissociation of PKA isoforms. (A–D) HEK293 cells were transfected with BRET sensors composed of mutant Renilla luciferase–conjugated R-subunits hRIα-RLuc8 (A), RIβ-RLuc8 (B), hRIIα-RLuc8 (C), and hRIIβ-RLuc8 (D) as bioluminescent donor proteins and GFP-tagged Cα-subunits as acceptor proteins. Close proximity of luciferase and GFP in the inactive PKA complex results in BRET signal (white), which is reduced after activation by Fsk/IBMX (gray; 50/100 µM, 20 min). Rp-8-Br-cAMPS (green), Rp-8-pCPT-cAMPS (blue), or Rp-8-Br-cAMPS (red) was applied 15 min before stimulation. Values are means ± SD; n = 3; one-way ANOVA with Bonferroni’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 indicate significance levels between control and Fsk-stimulated conditions; ^§, P < 0.05; ^§§, P < 0.01; ^§§§, P < 0.001 between Fsk-stimulations in the absence/presence of Rp-isomers; ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001 between the unstimulated controls in the absence/presence of Rp-isomers.