Extended Data Fig. 4: endo-GαsBP2 has a modest effect of cAMP responses, but efficiently blocks the ERK phosphorylation.

(A) endo-GαsBP2 minimally reduces GPCR-stimulated AC activity in cells, whereas mem-GαsBP2 efficiently blocks it. Diagram on the top left corresponds to the mechanism of cAMP regulation investigated with GαsBP2 probes. Graphs with kinetic traces on the top right correspond to luminescence-based cAMP measurements in HEK293T cells expressing 100 ng mem-GαsBP2 or 250 ng endo-GαsBP2 stimulated with isoproterenol as indicated. Mean ± S.E.M. (n = 4 independent experiments). The bar graph on the bottom left corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n = 4 independent experiments); ns, p > 0.05, **p < 0.01 by one-way ANOVA corrected for multiple comparisons (Dunnett). Immunoblots on the bottom right correspond to representative results of experiments confirming the expression of GαsBP2 constructs. The kinetic curve of the cAMP response from cells expressing mem-GαsBP2 is reproduced from Fig. 2e. (B) Endo-GαsBP2 efficiently blocks the GPCR-stimulated ERK phosphorylation. Diagram on the left corresponds the mechanism of ERK1/2 regulation investigated with GαsBP2 probes. ERK1/2 phosphorylation was detected in HEK293T cells expressing endo-GαsBP2 or mem-GαsBP2 were stimulated (or not) with 10 µM isoproterenol for 5 min, as indicated. A representative immunoblotting result is shown in the middle and the quantification of ERK1/2 phosphorylation is shown on the bar graph on the right. Mean ± S.E.M. (n = 5 independent experiments). *p < 0.05 by one-way ANOVA corrected for multiple comparisons (Dunnett).

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