Pglosensor 20f camp plasmid

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.

HEK293 cells stably expressing pGloSensor-20F cAMP plasmid (Promega) under Hygromycin selection were transfected with mCherry-2A-MC4R or the truncation mutants in wells of a 6 well dish. The next day cells were re-plated in 12 wells of a white bottom 96 well dish (for cAMP assay) and wells of a clear bottom plate (to determine expression). The following day, the media was removed from the clear bottom dish and replaced with Imaging buffer and the fluorescence was read on a Spectramax M3 plate reader (Molecular Devices). Fluorescence from empty-vector transfected cells was subtracted from each group and then the values were normalized to the fluorescence reading for the wild-type mCherry-2A-MC4R from each experiment. Data is from 3 independent experiments (mean ± SEM). For the cAMP assay, the media was carefully removed from the white-bottom plate and replaced with media containing 2% GloSensor cAMP reagent (Promega) and incubated at 37 °C for 2 hours. Cells were stimulated with various concentrations of α-MSH for 10 min and then the luminescence was read. Basal cAMP luminescence was subtracted, and cAMP values plotted as a percentage of maximum wildtype cAMP. Data is from 3 independent experiments (mean ± SEM).