*4.4. Immunocytochemistry*

Live-cell surface staining was performed as described previously [38]. The primary antibody used was mouse anti-rhodopsin 4D2 (Merck-Millipore). The secondary antibodies used were Cy3-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). After antibody staining, the cells were fixed in 4% paraformaldehyde, and then the slides were mounted with Mowiol and visualized by a fluorescence microscopy M1 imager (Carl Zeiss, Oberkochen, Deutschland).

#### *4.5. Dual Luciferase Reporter Gene Assay*

All odorants were purchased from FUJIFILM Wako Chemicals (Osaka, Japan) and TCI chemicals (Tokyo, Japan). The Dual-Glo Luciferase Assay (Promega) was used to determine the activities of firefly and Renilla luciferase in Hana3A cells as previously described [38]. Briefly, firefly luciferase, driven by a cAMP response element promoter (CRE-Luc; Stratagene), was used to measure the OR activation levels. For each well of a 96-well plate, 5 ng SV40-RL, 10 ng CRE-Luc, 5 ng mouse RTP1, 2.5 ng M3 receptor 3, and 5 ng of Rho-tagged receptor plasmid DNA were transfected. Normalized activity for each

well was further calculated as (Luc)/(Rluc). Luc and Rluc are the luminescence of firefly luciferase and Renilla luminescence, respectively. The basal activity was averaged from six wells in the absence of odorants and further corrected by subtracting that of the control empty vector. Odorant-induced activity was averaged from at least three wells and further corrected by subtracting the basal activity of that receptor. Odorant-induced responses were normalized to that of WT. EC50 value was calculated using GraphPad Prism software and data shown in Supplementary Data S2.
