Centrifugation for 20 min at 10,500 rpm (13,000 ) inside the SS34 rotor of a refrigerated centrifuge (Sorvall RC-5B). Protein concentration in the clarified lysate was measured making use of BCA reagent (Thermo Fisher Scientific, Waltham, Massachusetts, United states) then Fps1-3xFLAG was immunoprecipitated from a volume of extract containing a total of 10 mg protein using 50 l of mouse anti-FLAG 654671-77-9 site antibody coupled-agarose resin (Sigma Aldrich) equilibrated in TNE+Triton+NP40. Binding was permitted to occur for 2 hr at 4 . The resin was then washed extensively with TNE+Triton+ NP-40 and the proteins remaining bound have been then resolved by SDS-PAGE and analyzed by immunoblotting with acceptable antibodies to detect both Fps1-3xFLAG and Rgc2-3xHA.AcknowledgementsThis function was supported by NIH Predoctoral Instruction Grant GM07232 along with a Predoctoral Fellowship in the UC Systemwide Cancer Research Coordinating Committee (to AM), by NIH Predoctoral Instruction Grant GM07232 (to KLL), by NIH R01 Research Grant GM21841 and Senior Investigator Award 11-0118 from the American Asthma Foundation (to JT). We thank Stefan Hohmann (Univ. of Goteborg, Sweden), David E Levin (Boston Univ., Boston, MA), and Ted Powers (Univ. of California, Davis) for generously providing strains, plasmids and reagents, Hugo Tapia (Koshland Lab, UC Berkeley) for valuable discussions and reagents for measuring intracellular glycerol, and Jesse Patterson plus the other members from the Thorner Lab for numerous analysis materials and thoughtful ideas.Added informationFundingFunder National Institute of Common Health-related Sciences (NIGMS) University of California Berkeley (University of California, Berkeley) Grant reference T32 GM07232 Author Alexander Muir, Kristin L Leskoske Alexander 1616391-87-7 site MuirPredoctoral FellowshipMuir et al. eLife 2015;4:e09336. DOI: 10.7554/eLife.ten ofResearch advance Funder National Institute of Common Health-related Sciences (NIGMS) Foundation from the American College of Allergy, Asthma Immunology (ACAAI Foundation) Grant reference R01 GM21841 Author Jeremy ThornerBiochemistry | Cell biologySenior Investigator Award 11-Jeremy ThornerThe funders had no part in study style, information collection and interpretation, or the decision to submit the function for publication.Author contributions AM, FMR, Conception and design and style, Acquisition of information, Evaluation and interpretation of data, Drafting or revising the post; GT, Conception and design, Acquisition of data, Drafting or revising the post; KLL, Acquisition of data, Drafting or revising the short article; JT, Conception and design, Evaluation and interpretation of data, Drafting or revising the articleAdditional filesSupplementary files Supplementary file 1. Yeast strains made use of in this study.DOI: 10.7554/eLife.09336.Supplementary file 2. Plasmids used within this study.DOI: 10.7554/eLife.09336.
Neuropeptides are essential regulators of behavior. They’re able to act as nearby neurotransmitters (Salio et al., 2006) or as tonic “gain controls” on neuronal activity to modify diverse aspects of organismal physiology which includes appetite, biological rhythms, aggression, and more (Marder, 2012; Taghert and Nitabach, 2012). Neuropeptide signaling also modulates nociception, the sensory perception of noxious stimuli. For instance, Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) both regulate nociception in mammals (Harrison and Geppetti, 2001; Seybold, 2009). Modulation of nociception happens following tissue damage, where the threshold that elicits aversive beha.