HEK293T cells were seeded onto 18-mm coverslips coated with poly-L-lysine and transiently transfected with OR constructs with or without accessory proteins RTP1s and Ric8b (Lipofectamine 2000, Invitrogen). Flag-tagged OR trafficking was assayed using surface immunofluorescence as previously described (Shepard et al., 2013 (link)). Briefly, live, non-permeabilized cells at 4°C were exposed to a rabbit polyclonal anti-Flag antibody (Sigma) in 0.1% BSA/PBS. This antibody will only detect the extracellular Flag epitope of those receptors that are functionally expressed on the plasma membrane. Subsequently, cells were washed, fixed with 4% paraformaldehyde, permeabilized (0.3% Triton X-100), and then exposed to a mouse monoclonal (M2) anti-Flag antibody (Sigma). As the external Flag epitope (surface Flag) is ‘blocked’ after binding to the polyclonal Flag antibody, the monoclonal Flag antibody detects only the internal population of ORs. Alexa Fluor fluorescent secondary antibodies (Thermo Fisher) were used to detect the localization of the polyclonal and monoclonal Flag-tags. Slides were imaged using the ZEISS Axiovert 200 m microscope at 40× magnification.
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