Bombyx Neuropeptide GPCRs Figure 6. Expression of the AT receptor in sNPF-producing CC cells. (A) Expression analysis of 6 BNGRs within CC-CA complex on the fourth instar head capsule slippage. Vertical bars represent S.E. n = 3. Arbitrary unit is the same to Figure 3. (B) Expression analysis of sNPF on the fourth instar head capsule slippage. Relative expression was shown as a percentage of the expression level in the CC. For tissue names, see Figure 3. Inset, in situ hybridization of sNPF. Scale bar, 100 mm. (C) Double staining of the CC-CA with immunohistochemistry for sNPF (upper) and in situ hybridization for BNGR-A16 (lower). Scale bar, 100 mm. (D) Schematic illustration of the regulatory pathways of JH biosynthesis identified in this study. Putative role of AT in the regulation of sNPF production in the CC remains to be elucidated. doi:10.1371/journal.pone.0003048.g006 HEK293 cell expression and Ca2+ imaging analysis of BNGRs BNGRs cloned into pME18S vector were transfected into HEK293 cells with the promiscuous G protein Ga15 as described [53]. Ca2+ imaging analysis was performed as reported [53]. Briefly, the transfected cells were cultured for 24 h, and loaded with 2.5 mM fura-2 AM (Invitrogen, Carlsbad, CA) for 30 min at 37uC. Neuropeptides were applied sequentially to the cells for 20 s with a peristaltic pump at a flow rate of 1.5 ml/min. Fluorescence at 510 nm (excitation at 340 or 380 nm with a xenon lamp) was monitored with an intensified CCD camera. An Argus-HiSCA calcium-imaging system (Hamamatsu Photonics, Shizuoka, Japan) was used to determine the fluorescence ratio at 340 and 380 nm. In vitro radiochemical assay for JH biosynthesis JH biosynthesis by the CA was measured using an in vitro radiochemical assay as previously described [54], except that the CA was used instead of the CC-CA complex. The mixture of three sNPFs (1 mM each) was added to the incubation medium. Supporting Information Table S1 Summary of the two-way screening of Bombyx whole- genome shotgun sequence contigs. Query Drosophila GPCRs are aligned at the top, and the 195 contigs obtained through the first screening were listed on the left. Figures in the table indicate the BLAST scores of the first screening. Right two columns show the result of the second screening, which extracted 139 putative BNGR contigs (red). Found at: doi:10.1371/journal.pone.0003048.s001 (0.06 MB XLS) Table S2 List of contigs containing the fragments of previously reported Bombyx neuropeptide GPCR genes. Putative BNGR contigs are in red letters. Found at: doi:10.1371/journal.pone.0003048.s002 (0.02 MB XLS) Table S3 List of the newly-identified GPCRs and their corresponding contigs. Putative BNGR contigs are in red letters. Tissue sources for the cDNA clones are indicated on the right: FB, fat body; BR, brain; MG, midgut; OV, ovary; TE, testis; PG, prothoracic gland. Found at: doi:10.1371/journal.pone.0003048.s003 (0.02 MB XLS) Table S4 List of the primers used for the RACE of BNGRs. Found at: doi:10.1371/journal.pone.0003048.s004 (0.03 MB XLS) Table S5 List of the primers used for cloning of BNGRs. Each primer contains a restriction site indicated in the primer name, while all the sense primers incorporate Kozak sequence (‘‘GCCACC’’). Found at: doi:10.1371/journal.pone.0003048.s005 (0.02 MB XLS) PLoS ONE | www.plosone.org 10 August 2008 | Volume 3 | Issue 8 | e3048