2000;12(9):3239C3249. circuitry in the retina. Dimethyl trisulfide Here we display the localization of diacylglycerol lipase and (DGL/), implicated in the production of the eCB 2-arachidonoyl glycerol (2-AG); monoacylglycerol lipase (MGL) and /-hydrolase website 6 (ABHD6), both implicated in the breakdown of 2-AG; cannabinoid receptor interacting protein 1a (CRIP1a), a protein that may modulate CB1 function; Fatty acid amide hydrolase (FAAH) and (Bracey et al., 2002; Patricelli et al., 1998). Table 1 Antibodies used in this study. varieties, mono- vs. polyclonalrabbit polyclonal1:400mABHD6ABHD6-GST fusion protein, aa 104-141 of mouse ABHD6Mackie lab (Straiker et al., 2009),rabbit polyclonal1:1000rNAAANAAA-GST fusion protein, aa 261-275 of rat NAAAMackie lab (characterized in thisstudy), rabbit polyclonal1:400rDGLDGL-GST fusion protein, aa 790-908 of rat DGLMackie lab (Berghuis et al., 2007),guinea pig polyclonal1:600rDGLDGL-GST fusion protein, aa 790-908 of rat DGLMackie lab (Berghuis et al., 2007),rabbit polyclonal1:600rDGLDGL-GST fusion protein, aa 203-283 of rat DGLMackie lab (Berghuis et al., 2007),rabbit polyclonal1:600rCB1-L15CB1-GST fusion protein, aa 460-473 of rat CB1Mackie lab (Bodor et al., 2005),rabbit polyclonal1:1000rCB1-CTCB1-GST fusion protein, aa 401-473 of rat CB1Mackie lab (Hjos et al., 2000),rabbit polyclonal1:1000hCRIP1aCRIP1a-GST fusion protein, full-length protein of humanCRIP1aMackie lab (characterized in thisstudy), rabbit polyclonal1:400FAAHTM-FAAH which was raised against the purifiedtransmembrane-deleted FAAH-GST fusion protein, aa 38-579 ofrat FAAHCravatt lab (Bracey et al., 2002;Patricelli et al., 1998), rabbitpolyclonal1:100CalbindinPurified bovine kidney calbindin-D28KSigma-Aldrich, St. Louis, MO,#C9848, mouse monoclonal1:2000ParvalbuminPurified frog muscle mass parvalbuminSigma-Aldrich, St. Louis, MO,#P3088, mouse monoclonal1:1000GAD65Affinity-purified glutamic acid decarboxylase (GAD65) from rat brainDevelopmental StudiesHybridoma Standard bank, Iowa City, IA,mouse monoclonal1:600SV2Synaptic vesicles purified from your electrical organDevelopmental StudiesHybridoma Standard bank, Iowa City, IA,mouse monoclonal1:2000MAP2Microtubule-associated protein 2 (MAP2) purified from rat brainMillipore, Temecula, CA,#MAB3418, mouse polyclonal1:1000PKC (, 1, 2)PKC purified from bovine mind, and reacts with PKC , 1, and2 isoformsBioDesign, Saco, ME,#K01107M, mouse monoclonal1:200PSD95Purified recombinant rat PSD-95 (Post Synaptic Denseness 95 kDa)Genetex, Irvine, CA, #GTX80682,mouse monoclonal1:1000RecoverinRecombinant human being recoverin proteinChemicon, Temecula, CA,#Abdominal5585, rabbit polyclonal1:2000G13Synthetic peptide aa 47C59 of mouse G13Dr. Zaza Kokrashvili, Mount SinaiSchool of Medicine, New YorkCity, NY, rabbit polyclonal1:200NK3RSynthetic peptide of the C-terminus of rat NK-3 (aa 438C452)conjugated Dimethyl trisulfide to bovine thyroglobulinAbcam, Cambridge, MA, #ab7,rabbit polyclonal1:2000 Open in a separate window For the current study, we generated rabbit polyclonal antibodies for rat NAAA (rNAAA) and human being CRIP1a (hCRIP1a) and characterized their specificity as explained below. For NAAA, four GST fusion protein expression constructs were produced by inserting the DNA coding for four portions of rat NAAA (rNAAA) (designated like a, B, C, and D and corresponding to the following peptides: QDSQGRIYHGRNLD, SPHKFTISGDERDK, EGVVITRDRGGPAD, TNYDHWEPVPKRDD) into the pGEX-3X vector in the BamH I and EcoR I restriction sites. Each fusion protein was Rabbit polyclonal to AVEN purified from BL21 E. coli lysates on a glutathione sepharose column and the cocktail combination was injected into two rabbits to generate antisera (Cocalico Biologicals, Inc., Reamstown, PA) using standard methods (Bodor et al., 2005). The antiserum was purified in two methods, 1st by exclusion on a GST column and then by binding to and elution from an affinity column made with the rNAAA fusion protein C. For CRIP1a, a GST fusion protein expression construct was produced by inserting the DNA coding for full-length (MGDLPGLVRLSIALRIQPNDGPVFYKVDGQRFGQNRTIKLLTGSSYKVEVKIKPSTLQVENISIGGVLVPLELKSKEPDGDRVVYTGTYDTEGVTPTKSGERQPIQITMPFTDIGTFETVWQVKFYNYHKRDHCQWGSPFSVIEYECKPNETRSLMWVNKESFL) hCRIP1a into a pGEX-3X vector in the BamH I and EcoR I restriction sites. The fusion protein was purified from BL21 E. coli lysates on a glutathione sepharose column and was injected into two rabbits to generate antisera (R & R Rabbitry, Sedro-Wooley, WA) using standard methods (Bodor et al., 2005). The antiserum was purified in two methods, 1st by exclusion on a GST column and then by binding to and elution from an affinity column made with the hCRIP1a GST fusion protein. The Dimethyl trisulfide specificity of CB1-L15, CB1-CT, and FAAH have been previously characterized by using knockout mouse models and the immunostainings for CB1 and FAAH were completely absent in their related knockout (Bodor et al., 2005; Bracey et al., 2002; Hajos et al., 2000). In addition, the specificity of.