Measuring cAMP Response of Lamprey bPPL

The changes in the intracellular cAMP concentration of pigment-expressing HEK293S cells were measured using the GloSensor cAMP assay (Promega), as previously reported^{13 (link),29 (link)}. Briefly, cDNAs of lamprey bPPL were tagged with the epitope sequence for the monoclonal Rho1D4 antibody (ETSQVAPA). Tagged cDNA was inserted into the plasmid vector, pcDNA3.1 (Invitrogen). The expression constructs for bPPL were co-transfected with the pGloSensor-22F cAMP plasmid (Promega), and the transfected cells were incubated overnight in culture medium containing 10% fetal bovine serum (FBS) with 11-cis-retinal. Before the measurement, culture medium was replaced with a CO₂-independent medium containing 10% FBS and 2% GloSensor cAMP Reagent (Promega). After equilibration with the medium and obtention of a steady basal signal, the cells were treated with 3.5 μM forskolin, a direct activator of adenylyl cyclase, to increase the intracellular cAMP levels. Luminescence, representing the amount of cAMP, was measured at 25 °C using a GloMax 20/20n Luminometer (Promega). To measure the light-induced change in cAMP levels, the transfected cells were irradiated with blue LED light for 5 sec. The relative response curve of lamprey bPPL was analysed using the GloSensor cAMP assay without forskolin following the previously described method^{29 (link)}.