To generate the Obp49aD allele, the Obp49a1 flies www.selleckchem.com/products/ink128.html were crossed to flies containing the P[w+,Cre] transgene. The mosaic-eyed progeny were collected and crossed to balancer flies, and the white-eyed flies progeny of the latter cross were subjective to genomic PCR analysis using primers P1 and P3. To generate the UAS-Obp49a-t transgenic flies, we first amplified the Obp49a coding sequence lacking the translation stop codon from w1118

labellar complementary DNA (cDNA) using the High Fidelity PCR kit (Roche), and cloned the cDNA into the pUAST vector. Sequences encoding the 10 aa MYC linker (EQKLISEEDL) and the transmembrane domain from the platelet-derived growth factor receptor were amplified from the pDisplay vector (Invitrogen), and cloned in-frame 3′ to the coding region for Obp49a. We also subcloned the cDNA encoding Obp49a with a normal stop codon and without the sequences encoding MYC and the membrane-tethered tag (UAS-Obp49a)

into the pUAST vector. The transgenic flies were generated by BestGene. We extracted total RNA from the labella of adult male and female wild-type and poxn flies using the Trizol reagent (Invitrogen), and generated cDNAs from 0.5 μg RNA using the SuperScript III First Strand Synthesis

System http://www.selleckchem.com/products/pexidartinib-plx3397.html (Invitrogen). Quantitative PCR was performed using an ABI7500 real-time PCR machine (Applied Biosystems) and the ABI SYBR Green system. Transcript levels were normalized to rp49 as an internal control, and the ΔΔCT (CT = threshold cycle) method was used to calculate the relative why amount of mRNAs. We repeated the experiments at least four times. Rabbit polyclonal OBP49a antibodies were raised to a synthetic peptide (CKPPRGPPPSAEDM; amino acids 199–212). Twenty labella were dissected from wild-type, Obp49a1, and Obp49aD flies, and homogenized in 1× SDS sample buffer with pellet pestles (Kimble-Kontes). The extracts were subjected to electrophoresis by SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were probed with primary antibodies against OBP49a (1:1,000) and tubulin (1:3,000, 12G10 from Hybridoma Bank), and then with peroxidase-conjugated anti-mouse or rabbit IgG secondary antibodies (1:5,000; Sigma). Whole-mount fly labellar immunostaining was performed as described previously (Moon et al., 2009) using anti-OBP49a (1:400) and mouse anti-GFP (1:400; Molecular Probes) primary antibodies, and anti-mouse-Alexa488 (1:400; Molecular Probes) and anti-rabbit-Alexa568 (1:400; Molecular Probes) secondary antibodies.