Fig. 6
Nb5 as a control-switch for GPCR-mediated Gβγ signaling. a Schematic diagram of the GPCR-mediated Gβγ signaling network that controls various cellular functions. b Parental CHO-APJ cells, CHO-APJ cells transfected with pcDNA3.1 (+) empty vector, transfected CHO-APJ-Nb5 and CHO-APJ-Nb17 cells were treated with 1 μM apelin over 0–5 min to investigate the effect of Nb5 on the phosphorylation of ERK1/2 and AKT. Nb5 significantly decreased the phosphorylation of both ERK1/2 and AKT as compared to the Nb17 control (compare lanes marked with asterisks). Full blots are reported in Supplementary Figure 4b. c No significant effect of Nb17 transfection (purple) was observed on the phosphorylation of either ERK1/2 or AKT as compared to parental CHO-APJ cells (black) and CHO-APJ cells transfected with pcDNA3.1 (+) empty vector (red). Results are expressed as the mean ± SEM, n = 3 replicates, *P < 0.05, Student’s t-test. d Schematic diagram showing the effect of Gαi and Gβγ signaling on cellular cAMP levels. e Dose-response curves of apelin-induced inhibition of cAMP accumulation in parental CHO-APJ cells (grey) and CHO-APJ cells expressing either Nb5 (greencyan) or Nb17 (purple). Apelin concentration is plotted on a semi-log scale. Results are expressed as the mean ± SEM, n = 4 replicates. f Schematic diagram of GPCR-mediated second-messenger regulation by Gαq and Gαs. g The effect of nanobodies on Gαq signaling events was determined in HEK293T/17 cells transfected with M3R and CalFluxVTN. Each trace represents the mean of the responses measured in six wells. h Basal BRET ratio and ACh-induced maximum amplitude shown as a bar graph. Similar results were obtained in three independent experiments. Results are expressed as the mean ± SEM, n = 6 replicates. Two-way ANOVA with Tukey’s post hoc multiple comparison test. i Effect of nanobodies on Gαs signaling was evaluated in HEK293T/17 cells transfected with D1R and Nluc-Epac-VV. Each trace represents the mean of the responses measured in six wells. j Basal BRET ratio and dopamine-induced maximum amplitude shown as a bar graph. Each bar represents the mean of three independent experiments. Results are expressed as the mean ± SEM, n = 6 replicates. Two-way ANOVA with Tukey’s post hoc multiple comparison test
