Bombyx Neuropeptide GPCRs Figure 5. Reverse-physiological identification of sNPFs as novel allatostatic peptides. (A) Purification of sNPFs (arrowheads). The first (upper panel) and the fourth (lower two panels) HPLC separation steps are shown. Solid lines denote UV absorbance, while dashed lines denote the acetonitrile gradient. Fraction No. 22 in the upper panel was originally subjected to purification of BMS, and FMRFamide-related peptide (FaRP) content in this fraction was presented as the amount of purified BMS. (B) The deduced amino acid sequence of the sNPF precursor. The putative site of the signal peptide cleavage is indicated with an arrowhead, and the mature peptides are highlighted. For each mature peptide, C-terminal glycine residue is necessary for amidation. (C) Ca2+ imaging analysis using HEK293 cells expressing BNGR-A10. Relative response was expressed as a percentage of the highest response (induced by 1028 M sNPF-1–3). For each ligand, each datum point was calculated from the responses of 80– 122 cells in three independent experiments. (D) The effect of sNPF mixture (1 mM each) on JH biosynthesis by the fifth instar day 0 CA. Vertical bars represent S.E. n = 6 repeats. ***P,0.001 from Student’s t-test. doi:10.1371/journal.pone.0003048.g005 CAGCTCATCCATCTCG-39. Based on the obtained sequence of the 59-RACE product, 39-RACE was further performed using the following two primers: sense primer, 59-GATCGCCATCCC- GACGCCTGCGTTTCG-39; sense nested primer, 59-TGCG- TTTCGGCCGCCGATCAGATCCCG-39. First-strand cDNA prepared from larval brain total RNA and the SMART RACE cDNA Amplification Kit were used. Q-RT-PCR analysis of sNPF Q-RT-PCR of sNPF (GenBank accession no. AB330419) was performed as described above for the neuropeptide GPCR genes. Specific primers used are as follows: sense primer, 59-AAGT- CTGTTCGATCGCCATCCCGAC-39; antisense primer, 59-GA- GAGAGAGCAGCTCATCCATCTCG-39. The transcript levels of the sNPF were normalized with Rp49 levels in the same samples. In situ hybridization and immunohistochemistry In situ hybridization and immunohistochemistry was performed as previously described [9,12]. Specific primers used for the production of a digoxigenin-labeled probe were as follows: sNPF sense primer, 59-GGAGGGCTCTATGCTCTTCT-39; sNPF antisense primer, 59-TACATCTAGGTTGCGACGAG-39; BNGR-A16 sense primer 1, 59-GAGAATGAAACCTGCGTTG- GAGATCC-39; BNGR-A16 sense primer 2, 59-ACGCTGACT- TTCATCTCTGTGGACCG-39; BNGR-A16 antisense primer 1, 59-TGGTATGTCCTGGCTTGAAGGATGCC-39; BNGR-A16 antisense primer 2, 59-ACGCAGTTGTGTCAGTTCTGCAGT- GG-39; BNGR-A6-A sense primer 1, 59-CGCGAGAGCTAGAA- AACAGG-39; BNGR-A6-A sense primer 2, 59-CCGTCGTGAAT- CAATGTCTG-39; BNGR-A6-A antisense primer 1, 59-TCTTT- CCGAATCAACGCTCT-39; BNGR-A6-A antisense primer 2, 59- GCCACAACGGAATCAACTTT-39. For sNPF immunohisto- chemistry, rabbit polyclonal antiserum to RFamide [12] was used. Direct MS analysis of CC-CA CC-CA complex was dissected in sterile saline [0.85% NaCl (w/v)], and direct MS analysis was performed as previously described [12]. Synthetic peptides AST-C, AT, BMS, BRFa-3 and sNPF peptides were all custom synthesized and purified by HPLC. Synthetic AST-C was converted to the disulfide form by iodine oxidation and further purified by HPLC. PLoS ONE | www.plosone.org 9 August 2008 | Volume 3 | Issue 8 | e3048