Bombyx Neuropeptide GPCRs Figure 4. Functional characterization of CC-CA-expressed receptors. (A–C) Ca2+ imaging analysis using HEK293 cells expressing BNGR-A1 (A), BNGR-A16 (B) and BNGR-A6-A (C). Dose-response curves for AST-C (A), AT (B), ETH and pre- ecdysis-triggering hormone (PETH; C) are shown. Vertical bars represent S.E. Each datum point was calculated from the responses of 123-165 cells in three independent experiments. (D) Summary of the ligands for the CC-CA-expressed BNGRs analyzed by Ca2+ imaging analysis. doi:10.1371/journal.pone.0003048.g004 GACCGTGGAGGG-39; sense nested primer, 59-CCGGCAGT- GCTATTTCAATCCCATCTCC-39; antisense primer, 59-ATA- GGGCGAAAGCCGGGGTAAAAGTCG-39; antisense nested primer, 59-GGGTAAAAGTCGTGTTATTCACTTGCGG-39. To obtain the whole cDNA sequence of AST-C (GenBank accession no. AB330418), RACE was performed by using the above primers and the SMART RACE cDNA Amplification Kit. First-strand cDNA prepared from larval brain total RNA was used as a template. Purification of sNPF-2 and 3 Residual fractions of the first purification step of Bombyx pupal brain extracts, which were previously used for the purification of BMS [11], were applied to the FMRFamide ELISA as previously described [12]. One of the immunoreactive fractions (No. 16 in Figure 5A) was loaded onto a PEGASIL-300 C4P column (4.66250 mm, Senshu Kagaku Co. Ltd., Tokyo, Japan) using a Waters 2695 Separations Module (Waters, Milford, MA). Elution was performed with a linear gradient of 10–40% acetonitrile in 0.1% trifluoroacetic acid (TFA) over 60 min at the flow rate of 1 ml/min. This resulted in the detection of two FMRFamide-positive fractions, each of which was then applied onto a PEGASIL-300 ODS-II column (4.66250 mm, Senshu Kagaku Co. Ltd.). The column was eluted with a linear gradient of 10–40% acetonitrile in 0.1% TFA over 60 min at a flow rate of 0.5 ml/min. Each immunoreactive fraction was further purified by passing over a PEGASIL-300 ODS- II column (4.66250 mm) eluting it with a linear gradient of 10–40% acetonitrile in 0.1% heptafluorobutyric acid over 60 min at the flow rate of 0.5 ml/min. MALDI-TOF MS Mass spectra and postsource decay (PSD) mass spectra of peptides were obtained with a AXIMA-CFR (Shimadzu Corporation, Kyoto, Japan) mass spectrometer. Re-crystallized a-cyano-4-hydroxycin- namic acid (a-CHCA) was used as the matrix with a concentration of 5 mg/ml of acetonitrile/water (1:1, by volume) containing 0.1% TFA. All spectra were measured in reflector mode. Cloning of sNPF Using the amino acid sequence of one of the purified peptide [TPVR(L/I)RFG] as a query, BLAST analysis (TBLASTN) was performed against Bombyx whole-genome shotgun sequence contigs using KAIKOBLAST. Based on the putative coding sequence of the obtained contig (contig 432136, GenBank accession no. BAAB01085626), 59-RACE was performed using the following primer: antisense primer, 59-CGGAGAGAGAG- PLoS ONE | www.plosone.org 8 August 2008 | Volume 3 | Issue 8 | e3048