Mouse anti rhodopsin

HEK293T cells were plated on poly-D-lysine-coated 96-well plates (Greiner). Plasmid DNA of ORs containing Rho tag and accessory proteins were transfected using Lipofectamine2000 (Invitrogen). For each 96-well plate, 3.2 μg of OR and 6.4 μg total of all accessory proteins (RTP1S and/or RTP2) or pCI vector as a negative control were transfected. For example, 3.2 μg of one accessory protein and 3.2 μg of pCI were transfected to make the total amount of plasmids constant. 16–18 hr post-transfection, cells were fixed in 4% paraformaldehyde for 15 min and blocked in 5% FBS/0.5% BSA diluted in phosphate-buffered saline at 4 °C. The cells were then incubated in 5% FBS/0.5% BSA diluted in phosphate-buffered saline containing the primary antibody mouse anti-rhodopsin (Millipore) at 4 °C for 1 hr. The cells were then washed in phosphate-buffered saline followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Beyotime) at 4 °C for 45 min. The signals were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore) according to the manufacturer’s instructions and luminescence was measured using the Infinite F200 Pro plate reader (Tecan). Normalized luminescence values for quantifying the OR cell-surface expression were calculated by the formula OR/pCI. Data were analyzed with Microsoft Excel and GraphPad Prism 5.