Pcmv6 entry c terminal myc and ddk tagged

HMEC-1 WT cells were transfected with pCMV6-Entry (C-terminal Myc and DDK Tagged, OriGene Technologies, Rockville, MD) or OPN4 (Myc-DDK-Tagged)-pCMV6-Entry transcript variant 1 (TrueORF Gold Expression-Validated cDNA Clones from OriGene Technologies, Rockville, MD) using FuGene HD Transfection Reagent (Promega, Madison, WI). After transfection, cells were kept in complete darkness until room light (~200 LUX) or intense light (~10,000 LUX) exposures for 30 minutes. Melanopsin protein expression validation was done by isolating protein using RIPA buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Immunoblotting for anti-DDK (OriGene Technologies, Rockville, MD) was used to detect DDK-tagged melanopsin in transfected cells. Light-sensing cells subjected to glycolytic or mitochondrial stress tests on the Seahorse Bioanalyzer were exposed to light 30 min prior to Seahorse analyses.

Transfection was performed in 6-well plates with FuGENE 6 transfection reagent (Promega, USA, E2691) according to the manufacturer’s instructions. 100 μl of DMEM serum-free medium containing 2μg of vector DNA: LXN (Myc-DDK-tagged)-Human LXN (LXN) (Origene, RC202769) or pCMV6-Entry (C-terminal Myc and DDK Tagged) (Origene, PS100001) and 3 μl of FuGENE reagent was incubated for 15 minutes at room temperature before the mixture was added in 6 cm-well cell cultures (50% confluent) in the presence of 10% fetal bovine serum. After transfection for 48h, the cells were treated with 50–200ug/ml G418 for selection of transfected cells.