Functional Expression of Olfactory Receptors

MDCK cells were grown on 12 mm Transwell polycarbonate membranes (0.4 μm pore; Sigma) and transfected with the Lucy-Flag-Olfr1393 construct that was described previously57 (link) along with RTP1S59 (link) (modified from RTP1L). The cleavable Lucy tag was used to promote functional expression of ORs in heterologous cells. After 24 h, the media was replaced and the cells were allowed to grow to confluency and polarize (72 h). To label the apical membrane, the top side of the Transwell membranes containing live, non-permeabilized cells was exposed to a rabbit polyclonal anti-Flag antibody (Sigma) at 4 °C. Subsequently, the cells were washed, fixed with 4% paraformaldehyde, permeabilized (0.3% Triton X-100) and exposed to a mouse gp135 antibody26 . To detect total Flag-Olfr1393, the live cell labeling steps were skipped and the cells were immediately fixed and permeabilized and probed for Flag and gp135. The confocal images were taken with a Zeiss AxioObserver LSM700 and Zen software.