Researchers discover a new way to develop drugs without sideeffects nature
activation was measured using the GloSensor cAMP accumulation assay. We first constructed a plasmid wherein the human full-lengthgene was N-terminally fused to the Flag epitope tag with the preceding haemagglutinin-derived signal sequence. HEK293A cells were seeded in a 6-cm culture dish at a concentration of 2 × 10cells per ml supplemented with 10% FBS and penicillin–streptomycin–glutamine ) 1 day before transfection.
The luminescence counts over 8–10 min after ligand addition were averaged and normalized to the initial counts, and the fold changes in the signals over the vehicle treatment were further normalized to forskolin and plotted for the cAMP accumulation response. Using the Prism 9 software , the response were fitted to all data using the nonlinear regression. The variable slope in the Prism 9 tool with a constraint of the Hill slope of absolute value less than 2 was used.
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