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Supporting Information Supporting Information Blankman et al. 10.1073/pnas.1217121110 SI Materials and Methods RT-PCR. Total RNA was isolated from brain tissue of 6-wk-old −/− +/+ Materials. We purchased 2-arachidonoylglycerol (2-AG) from ABHD12 and ABHD12 mice using TRIzol (Invitrogen). Cayman Chemicals; 2-oleoylglycerol, pentadecanoic acid (PDA), First-strand cDNA was synthesized using the SuperScript III and dodecylmonoalkylglycerol ether (C12:0 MAGE) were pur- Reverse Transcriptase kit (Invitrogen) according to the manu- chased from Sigma-Aldrich. Monopentadecanoin (C15:0 MAG) facturer’s protocol. PCR amplification (25 cycles) of a 259 bp fragment of the ABHD12 cDNA was performed with primers 50- and monoheptadecanoin (C17:0 MAG) were purchased from Nu- 0 0 Chek-Prep. Phospholipids and lysophospholipids were purchased CTCAGTAGGAACACCATCGGAGC-3 and 5 -GCCAGGG- AAGTATCGGTATATCACTG-3. Amplification of a 245-bp from Avanti Polar Lipids. Fluorophosphonate (FP)-rhodamine 0 (1) and JZL184 (2) were synthesized as described previously. GAPDH product was performed as a control with primers 5 - GGTGAAGGTCGGTGTGAACGG-30 and 50-CCCATTTGA- 0 Generation of ABHD12−/− Mice. The α/β-hydrolase domain-con- TGTTAGTGGGGTCTCG-3 . Both sets of primers were de- taining (ABHD)12-targeting construct was generated by ampli- signed to span a >2-kb region of genomic DNA. fying 3.4- and 4.4-kb regions of the Abhd12 gene adjacent to the Untargeted liquid chromatography–mass spectrometry proteomic analysis. catalytic exon 8 from a BAC clone containing the Abhd12 locus Mouse brain membrane proteomes were prepared from 6-mo-old − − + + (clone ID RP23-193L22 from BACPAC Resources Center at ABHD12 / and ABHD12 / mice (n = 3 per genotype). Mem- Children’s Hospital Oakland Research Institute) and subcloning brane proteins (0.375 mg of total protein) were precipitated with these homologous arms into the SacII/BamHI and XhoI/HindIII 1:4 (vol/vol) CHCl3:MeOH and denatured with 25 mM ammo- sites of the pKO-NTKV vector. The targeting construct was nium bicarbonate in 6 M urea. Samples were reduced with designed to replace Abhd12 exons 8–10 with a neomycin selec- 10 mM DTT, alkylated with 40 mM iodoacetamide, and diluted tion cassette upon homologous recombination. Following elec- to 2 M urea with 25 mM ammonium bicarbonate. Digestion with troporation of the targeting construct in C57BL/6 Bruce 4-derived trypsin (0.5 μg/μL) was performed overnight at 37 °C in the murine embryonic stem (ES) cells, 300 integrated ES cell clones presence of 1 mM CaCl2. The tryptic peptide samples were were obtained and screened for homologous recombination by acidified with 5% (vol/vol) formic acid, and aliquots were frozen Southern blot analysis with external probes located 50 and 30 of the at −80 °C until use. targeted region. These probes gave differential band sizes in WT Multidimensional protein identification technology (MudPIT) (10.4 kb) or targeted (50 probe, 4.8 kb; 30 probe, 5.7 kb) genomic analysis was performed as described previously (3) on an LTQ DNA digested with EcoRI. One homologous recombinant, 96, was Orbitrap Velos mass spectrometer (ThermoFinnigan) coupled to identified, expanded and injected into albino C57BL/6 blastocysts. an Agilent 1200 series HPLC (50 μg of protein; 11-step gradi- Blastocyst implantation into pseudopregnant albino C57BL/6 fe- ent). MS spectra were acquired in profile mode, with a mass males generated nine chimeric males. Three of the chimeras pro- range of 400–1,800 in the Orbitrap analyzer with resolution set at duced germ-line transmission of the targeted mutation, which was 30,000, followed by 30 MS/MS scans in the ion trap. Dynamic confirmed by Southern analysis. PCR genotyping of genomic tail exclusion was enabled with a repeat count of 1, a repeat duration DNA was performed using the primers 50-CAGTGCTGGCCT- of 20 s, exclusion duration 20 s, and an exclusion list size of 300. GTCAGTCG-30, 5-GGTGCCCAGTGAATGGCC-30,and50-TA- All tandem mass spectra were collected using a normalized AAGCGCATGCTCCAGACTGCC-30, which amplify a 467-bp collision energy of 35%, an isolation window of 2 Da, and an product from the WT allele and an 295-bp product from the activation time of 10 ms. One microscan was applied for all ex- targeted allele. All mice used in this study were generated from periments. Spray voltage was set to 2.50 kV, and the flow rate + − breeding ABHD12 / mice and had ad libitum access to water through the column was 0.20 μL/min. and food. RAW files were generated from the mass spectra using XCalibur version 1.4, and MS/MS spectra data extracted using Biochemical Studies. Preparation of mouse brain proteomes. Mice were RAW Xtractor (Version 1.9.1), which is publicly available anesthetized with isoflurane and killed by decapitation. Brains (http://fields.scripps.edu/?q=content/download). The tandem were harvested, sectioned into hemispheres, immediately flash MS data were searched against the mouse International Protein frozen in liquid nitrogen, and frozen at −80 °C until use. One half Index (IPI) database using the ProLuCID search algorithm (4), brain was Dounce-homogenized in PBS (pH 7.5), sonicated, and allowing for modification of M with the crosslinking agent centrifuged at slow speed (1,400 × g for 10 min at 4 °C) to re- (15.9949), static modification of cysteine residues (57.02146 Da, move debris. The supernatant was centrifuged at high speed because of alkylation), half-tryptic enzyme specificity, and a mass (100,000 × g for 45 min at 4 °C), and this supernatant was saved tolerance set to 50 ppm for precursor mass and ±0.6 Da for as the soluble proteome. The pellet was washed and resuspended product ion masses. The resulting MS/MS spectra matches were in PBS, sonicated, and saved as the membrane proteome. The assembled and filtered using DTASelect2 (Version 2.0.27). The total protein concentration of each proteome was determined validity of peptide/spectrum matches was assessed using DTA- using the Bio-Rad Dc Protein Assay kit. Aliquots of the pro- Select2 (Version 2.0.27) and two SEQUEST-defined parame- teomes were stored at −80 °C until use. ters: the cross-correlation score (XCorr) and the normalized Activity-based protein profiling analysis. Brain membrane proteomes difference in cross-correlation scores (DeltaCN). Peptides with (50 μgin50μL of PBS buffer) were prepared from 6-wk-old XCorr scores greater than 1.8 (+1), 2.5 (+2), or 3.5 (+3), and − − + − + + ABHD12 / ,ABHD12/ ,andABHD12/ littermates and pre- DeltaCN scores greater than 0.08 were included in the spectral treated with 5 μM JZL184 or DMSO vehicle for 30 min at 25 °C, counting analysis. followed by incubation with 2 μM FP-rhodamine (1) for 1 h at 25 °C. Lipid substrate hydrolysis assays. The 2-AG hydrolysis activity of − − + + Reactions were quenched with 2× SDS/PAGE loading buffer (re- brain membrane homogenates from ABHD12 / and ABHD12 / ducing), separated by SDS/PAGE [10% (wt/vol) acrylamide] and mice (2 mo old; n = 4 per genotype) plus 1 μM JZL184 or vehicle visualized in-gel with a FMBio IIe flatbed fluorescence scanner (DMSO) was measured as described previously (5). Assessing the (Hitachi). Rhodamine fluorescence is shown in gray scale. activity of brain membrane homogenates against C18:1 mono- Blankman et al. www.pnas.org/cgi/content/short/1217121110 1of19 acylglycerol (MAG), C18:1 lysophosphatidylserine (LPS), C18:1 The fatty acid or lysophospholipid hydrolysis products were lysophosphatidylinositol (LPI), C18:1 lysophosphatidylglycerol quantified by measuring the area under the peak in comparison (LPG), C18:1 lysophosphatidylcholine (LPC), C18:1 lysophos- with the internal standard and corrected for the background phatidic acid (LPA), and C18:1/18:1 bis(monoacylglcerol)phos- product present in reactions performed with heat inactivated (10 phate (BMP) was performed similarly. Mouse brain membrane min at 90 °C) protein homogenates. Data are presented as the − − proteomes were prepared from 6-mo-old ABHD12 / and mean of three replicates ± SEM. Statistical analysis was performed + + ABHD12 / littermates (n = 3 per genotype). Activity assays by Student’s t test. were performed with 20 μg of brain membrane homogenate di- luted in PBS (100 μL total volume) incubated with 100 μM lipid Behavioral Analysis. All behavioral testing, except for the tetrad substrate. After 30 min at 25 °C, the reactions were quenched tests for cannabimimetic activity, was performed in The Scripps with 2:1 (vol/vol) CHCl3:MeOH (350 μL), and 0.5 nmol PDA Research Institute mouse behavior assessment core facility. Mice was added as an internal standard. The reaction vials were vor- were group-housed in a temperature-controlled room in which texed to mix and centrifuged for 5 min at 1,400 × g to separate the lights were on a 12-h light/dark cycle (lights off at 0600 hours), phases. A portion of the lower organic phase (30 μL) was in- and behavior was assessed during the dark (active) phase. Food jected onto an Agilent 6520 series quadrupole–time-of-flight and water were available ad libitum. − − + − + + (QTOF) MS. Chromatography was performed on a 50 × 4.60 A cohort of ABHD12 / , ABHD12 / , and ABHD12 / lit- mm 5-μm Gemini C18 column (Phenomenex) coupled to a guard termates (n = 8–9 per genotype; mixed sex) were tested for lo- column (Gemini; C18; 4 × 3.0 mm; Phenomenex SecurityGuard comotor activity in an open-field, startle responses to auditory cartridge). The LC method consisted of 0.1 mL/min of 100% stimuli, rotarod performance, and hanging wire performance at – – – = – buffer A [95:5 (vol/vol) H2O:MeOH plus 0.1% (vol/vol) am- 5 6, 11 12, and 17 18 mo of age. A second cohort (n 8 10 per monium hydroxide] for 1.5 min, 0.5 mL/min linear gradient to genotype; mixed sex) was tested at 12 and 18 mo of age.
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