OBP49a was purified by serial use of HiTrap SP XL 5 ml and HiTrap Q XL 5 ml columns (GE Healthcare), followed click here by affinity purification with OBP49a antibodies. The purity of OBP49a was assessed by fractionation of the protein by SDS-PAGE and silver staining ( Figures S4C and S4D). SPR was conducted using a BIAcore 3000 (GE Healthcare)

at 25°C. Coupling of OBP49a to CM5 chips (GE Healthcare) was performed by injecting 0.1 μg/ml of protein with 10 mM sodium acetate, pH 4.0, at a 5 μl/min flow rate, and confirmed by an increase of 10,000 resonance units on the sensor chip. The chemicals were diluted to the indicated concentrations in continuous flow buffer (HBS-P [10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Surfactant P20]). Each analytic run was performed at a 30 μl/min flow rate. The chip matrix was regenerated using 20 mM NaOH after each binding analysis. To obtain the UAS-YFP(1):Gr64a, UAS-YFP(1):Gr64f transgenic flies for the PCA, we first generated pUAST-YFP(1) by PCR amplifying a 462 bp YFP(1) www.selleckchem.com/products/3-methyladenine.html fragment from pAKAR3EV ( Komatsu et al., 2011) that extended from the Kozak sequence. This fragment was subcloned between the EcoRI and KpnI sites of pUAST. We then inserted

the coding sequences of Gr64a and Gr64f into pUAST-YFP(1), so that YFP(1) was linked to the N termini of the GRs. To produce the pUAST-OBP49a-t-YFP(2) construct, we used pUAST-Obp49a-t to PCR amplify the OBP49a-t coding sequence that lacked the stop codon, and then inserted the fragment into pUAST. We then used pAKAR3EV as the template to PCR amplify two DNA fragments encoding a 116 amino acid long flexible EV linker and YFP(2), which encoded residues 155–237 of YFP. We inserted these DNA fragments adjacent to the 3′ end of the OBP49a-t coding region. We expressed these transgenes, as well as UAS-Snmp1-YFP(2) ( Benton et al., almost 2007), under the control of Gr5a-GAL4. To apply berberine to the sensilla, we immobilized the flies with a glass capillary and dipped the labella

into a solution containing 100 μM berberine for 1 min before dissecting the labella. We also immersed labella in 100 μM berberine/100 mM sucrose solutions, and obtained results indistinguishable from those generated with untreated labella or labella dipped in berberine only (data not shown). The labella were fixed with 4% paraformaldehyde in PBS-T (0.2% Triton X-100 in PBS) for 20 min. Fixed labella were washed with PBS-T three times, cut in half with a razor blade, and then mounted in VECTASHIELD (Vector Laboratories). Fluorescence was viewed in whole mounts of labella using a Zeiss LSM700 confocal microscope. All error bars represent SEMs. Unpaired Student’s t tests were used to compare two sets of data. ANOVA with Tukey post hoc tests were used to compare multiple sets of data. Asterisks indicate statistical significance (∗p < 0.05, ∗∗p < 0.01). We thank FlyBase, the Bloomington Stock Center and Drs. K. Scott, L. Vosshall, J.W. Posakony, and Y.D. Chung for fly stocks, and Dr. C.Y. Park and Mr. J.