Pcdna3.1 mit 2mutaeq

The intracellular cAMP and Ca²⁺ levels in rhodopsin-expressing HEK293S cells (human embryonic kidney 293 S cells, provided by Dr. Jeremy Nathans of Johns Hopkins University) were measured using the GloSensor cAMP assay and the aequorin assay, respectively, as described previously (Bailes and Lucas, 2013 (link)). HEK293S cells have been confirmed to be free from mycoplasma contamination. The identity of HEK293S cells was confirmed by similarity to HEK293 and HEK293T cells through STR profiling, and by morphological observation of the cells. The pGloSensor-20F cAMP plasmid (Promega) was used for the GloSensor cAMP assay. The wild type aequorin obtained by introducing two reverse mutations into the plasmid [pcDNA3.1+/mit-2mutAEQ] (Addgene #45539) (de la Fuente et al., 2012 (link)) was used for the aequorin assay. The rhodopsin expression plasmids were constructed based on pCS2+ (see the Zebrafish section) and used for transfection. For Gαq inhibition, YM-254890 (FUJIFILM Wako Pure Chemical Corp., 257–00631, Osaka, Japan) was added (1 μM) 5 min before the measurement. Green (500 nm) and violet (410 nm) LED lights were applied for 5 s in the GloSensor cAMP assay and for 1 s in the aequorin assay as light stimuli. Dual Head LED Light 505 nm (GB Life Science) and SPL-25-CC (REVOX, Inc) were used for green and violet LED light stimulation, respectively.