Journal: ACS Chemical Neuroscience

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

doi: 10.1021/cn300007s

Figure Lengend Snippet: Substrate specificity of NAAA in the presenceof different stimulators.Recombinant NAAA (0.3–3 μg of protein) was allowed toreact with the indicated 14 C-labeled NAEs at 100 μMin the presence or absence of 0.1% Nonidet P-40,

Article Snippet: [1-14 C]Palmitic acid was purchasedfrom PerkinElmer Life Science (Boston, MA); [1-14 C]stearicand [1-14 C]arachidonic acids from GE Healthcare (Piscataway,NJ); [1-14 C]oleic acid from Moravek Biochemicals (Brea,CA); nonradiolabeled NAEs from Cayman Chemical (Ann Arbor, MI); bovineserum albumin, 1,2-dioleoyl-PE, 1,2-dioleoyl-PC, 1,2-dipalmitoyl-PG,bovine liver PI, 1,2-dioleoyl-PS, bovine brain SM, 2-mercaptoethanol,dihydrolipoic and α-lipoic acids, and fetal calf serum fromSigma-Aldrich (St. Louis, MO); Triton X-100, Tween 20, DTT, l -cysteine hydrochloride, glutathione, formic and succinic acids,sodium (+)-tartrate, 3(2)- t -butyl-4-hydroxyanisole,ethanolamine, and Dulbecco’s modified Eagle’s mediumfrom Wako Pure Chemical (Osaka, Japan); Nonidet P-40, dl -homocysteine,potassium hydrogen phthalate, and l -(+)-tartaric and 3,3-dimethylglutaricacids from Nacalai Tesque (Kyoto, Japan); n -octyl-β- d -glucoside and CHAPS from Dojindo (Kumamoto, Japan); proteinassay dye reagent concentrate from Bio-Rad (Hercules, CA); precoatedsilica gel 60 F254 aluminum sheets for thin-layer chromatography(20 × 20 cm, 0.2-mm thickness) from Merck (Darmstadt, Germany);Lipofectamine 2000, TRIzol, and pcDNA3.1(+) from Life Technologies(Carlsbad, CA); PrimeScript RT reagent kit and SYBR Premix Ex Taq II from Takara Bio (Ohtsu, Japan); and HEK293 cells fromHealth Science Research Resources Bank (Osaka, Japan).

Techniques: Recombinant, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

doi: 10.1021/cn300007s

Figure Lengend Snippet: Effects of various buffers on NAAA activity. Recombinant NAAA (300ng of protein) was allowed to react with 100 μM [ 14 C]PEA in the presence of 3 mM DTT and 0.1% Nonidet P-40 at pH 4.5.The pH was adjusted with the following buffers (100 mM): citrate-Na

Article Snippet: [1-14 C]Palmitic acid was purchasedfrom PerkinElmer Life Science (Boston, MA); [1-14 C]stearicand [1-14 C]arachidonic acids from GE Healthcare (Piscataway,NJ); [1-14 C]oleic acid from Moravek Biochemicals (Brea,CA); nonradiolabeled NAEs from Cayman Chemical (Ann Arbor, MI); bovineserum albumin, 1,2-dioleoyl-PE, 1,2-dioleoyl-PC, 1,2-dipalmitoyl-PG,bovine liver PI, 1,2-dioleoyl-PS, bovine brain SM, 2-mercaptoethanol,dihydrolipoic and α-lipoic acids, and fetal calf serum fromSigma-Aldrich (St. Louis, MO); Triton X-100, Tween 20, DTT, l -cysteine hydrochloride, glutathione, formic and succinic acids,sodium (+)-tartrate, 3(2)- t -butyl-4-hydroxyanisole,ethanolamine, and Dulbecco’s modified Eagle’s mediumfrom Wako Pure Chemical (Osaka, Japan); Nonidet P-40, dl -homocysteine,potassium hydrogen phthalate, and l -(+)-tartaric and 3,3-dimethylglutaricacids from Nacalai Tesque (Kyoto, Japan); n -octyl-β- d -glucoside and CHAPS from Dojindo (Kumamoto, Japan); proteinassay dye reagent concentrate from Bio-Rad (Hercules, CA); precoatedsilica gel 60 F254 aluminum sheets for thin-layer chromatography(20 × 20 cm, 0.2-mm thickness) from Merck (Darmstadt, Germany);Lipofectamine 2000, TRIzol, and pcDNA3.1(+) from Life Technologies(Carlsbad, CA); PrimeScript RT reagent kit and SYBR Premix Ex Taq II from Takara Bio (Ohtsu, Japan); and HEK293 cells fromHealth Science Research Resources Bank (Osaka, Japan).

Techniques: Activity Assay, Recombinant

Journal: ACS Chemical Neuroscience

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

doi: 10.1021/cn300007s

Figure Lengend Snippet: Effects of various phospholipid classes on NAAA activity.RecombinantNAAA (300 ng of protein) was allowed to react with 100 μM [ 14 C]PEA. (A) The reactions were carried out in the presenceof 3 mM DTT and either Nonidet P-40 at 0.1% (w/v) or the

Article Snippet: [1-14 C]Palmitic acid was purchasedfrom PerkinElmer Life Science (Boston, MA); [1-14 C]stearicand [1-14 C]arachidonic acids from GE Healthcare (Piscataway,NJ); [1-14 C]oleic acid from Moravek Biochemicals (Brea,CA); nonradiolabeled NAEs from Cayman Chemical (Ann Arbor, MI); bovineserum albumin, 1,2-dioleoyl-PE, 1,2-dioleoyl-PC, 1,2-dipalmitoyl-PG,bovine liver PI, 1,2-dioleoyl-PS, bovine brain SM, 2-mercaptoethanol,dihydrolipoic and α-lipoic acids, and fetal calf serum fromSigma-Aldrich (St. Louis, MO); Triton X-100, Tween 20, DTT, l -cysteine hydrochloride, glutathione, formic and succinic acids,sodium (+)-tartrate, 3(2)- t -butyl-4-hydroxyanisole,ethanolamine, and Dulbecco’s modified Eagle’s mediumfrom Wako Pure Chemical (Osaka, Japan); Nonidet P-40, dl -homocysteine,potassium hydrogen phthalate, and l -(+)-tartaric and 3,3-dimethylglutaricacids from Nacalai Tesque (Kyoto, Japan); n -octyl-β- d -glucoside and CHAPS from Dojindo (Kumamoto, Japan); proteinassay dye reagent concentrate from Bio-Rad (Hercules, CA); precoatedsilica gel 60 F254 aluminum sheets for thin-layer chromatography(20 × 20 cm, 0.2-mm thickness) from Merck (Darmstadt, Germany);Lipofectamine 2000, TRIzol, and pcDNA3.1(+) from Life Technologies(Carlsbad, CA); PrimeScript RT reagent kit and SYBR Premix Ex Taq II from Takara Bio (Ohtsu, Japan); and HEK293 cells fromHealth Science Research Resources Bank (Osaka, Japan).

Techniques: Activity Assay

Journal: ACS Chemical Neuroscience

Article Title: Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

doi: 10.1021/cn300007s

Figure Lengend Snippet: Effects of various thiol compounds onNAAA activity. RecombinantNAAA (300 ng of protein) was allowed to react with 100 μM [ 14 C]PEA. (A) The reactions were carried out in the presenceof 0.1% Nonidet P-40 and the indicated compound at 3 mM. (B)

Article Snippet: [1-14 C]Palmitic acid was purchasedfrom PerkinElmer Life Science (Boston, MA); [1-14 C]stearicand [1-14 C]arachidonic acids from GE Healthcare (Piscataway,NJ); [1-14 C]oleic acid from Moravek Biochemicals (Brea,CA); nonradiolabeled NAEs from Cayman Chemical (Ann Arbor, MI); bovineserum albumin, 1,2-dioleoyl-PE, 1,2-dioleoyl-PC, 1,2-dipalmitoyl-PG,bovine liver PI, 1,2-dioleoyl-PS, bovine brain SM, 2-mercaptoethanol,dihydrolipoic and α-lipoic acids, and fetal calf serum fromSigma-Aldrich (St. Louis, MO); Triton X-100, Tween 20, DTT, l -cysteine hydrochloride, glutathione, formic and succinic acids,sodium (+)-tartrate, 3(2)- t -butyl-4-hydroxyanisole,ethanolamine, and Dulbecco’s modified Eagle’s mediumfrom Wako Pure Chemical (Osaka, Japan); Nonidet P-40, dl -homocysteine,potassium hydrogen phthalate, and l -(+)-tartaric and 3,3-dimethylglutaricacids from Nacalai Tesque (Kyoto, Japan); n -octyl-β- d -glucoside and CHAPS from Dojindo (Kumamoto, Japan); proteinassay dye reagent concentrate from Bio-Rad (Hercules, CA); precoatedsilica gel 60 F254 aluminum sheets for thin-layer chromatography(20 × 20 cm, 0.2-mm thickness) from Merck (Darmstadt, Germany);Lipofectamine 2000, TRIzol, and pcDNA3.1(+) from Life Technologies(Carlsbad, CA); PrimeScript RT reagent kit and SYBR Premix Ex Taq II from Takara Bio (Ohtsu, Japan); and HEK293 cells fromHealth Science Research Resources Bank (Osaka, Japan).

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose

doi: 10.1074/jbc.M808890200

Figure Lengend Snippet: PGC-1α interacts with OGT. A , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and OGT co-immunoprecipitation ( IP ). Immunoprecipitates were subjected to SDS-PAGE and immunoblotted ( IB ) for the presence of PGC-1α. Membranes were then stripped and blotted for OGT. Immunoblots are representative of three experiments. B , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and FLAG co-immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for the presence of OGT. Membranes were then stripped and blotted for PGC-1α. C , gel filtration chromatography (SMART system, Superdex 200 column, phosphate-buffered saline, 1% Nonidet P-40 buffer) of lysates from rat liver incubated on ice for 30 min with either normal IgG or anti-OGT antibodies (AL28). Fractions were subjected to SDS-PAGE and blotted using anti-PGC-1α, anti-OGT (DM-17), and anti CPB. Data are representative of two experiments.

Article Snippet: Rat liver was lysed in Tris-buffered saline with 1% Nonidet P-40 and incubated for 1 h on ice with either normal rabit IgG (Santa Cruz Biotechnology) or anti-Ogt (AL28) ( ).

Techniques: Pyrolysis Gas Chromatography, Infection, Immunoprecipitation, SDS Page, Western Blot, Filtration, Chromatography, Incubation