Transient Expression and Purification of T1r Proteins

Transient expression of T1rLBD proteins in S2 cells was carried out by the calcium phosphate method as described previously22 (link), or by using FuGENE HD (Roche) with 1 μg each T1r2aLBD and T1r3LBD expression vectors for 1 × 10⁶ cells according to the manufacturer's protocol. The cells were cultivated at 300 K for 4 days. Mutations were introduced using the QuikChange method (Agilent Technologies).
The cell culture supernatant was mixed with ANTI-FLAG M2 affinity gel (SIGMA) and rotated at 277 K. After washing with 20 mM Tris, pH 7.4, containing 150 mM NaCl, the proteins retained on the beads were eluted with 2 × SDS–PAGE sample buffer (100 mM Tris, 2% SDS, 10% (v/v) glycerol, 0.002% bromophenol blue, pH6.8), followed by heating at 368 K for 5 min. The eluents were divided into two equal parts, further incubated at 368 K for 5 min with or without 100 mM DTT, and subjected to SDS-PAGE followed by electrophoretic transfer onto membranes, using an iBlot apparatus (Life Technologies). T1rLBDs were detected using anti-DDDDK tag-HRP (1:2,000) (Cat. # PM020-7, Medical and Biological Laboratories) and Immobilon Western Chemiluminescent HRP Substrate (Millipore). The images were obtained using a ChemiDoc Imager (Bio-Rad). The uncropped original images of the blots are shown in Supplementary Fig. 9.