Recombinant:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
Real-time Polymerase Chain Reaction:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
Protease Inhibitor:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
ALP Assay:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
Labeling:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
Enzyme-linked Immunosorbent Assay:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
Reporter Assay:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
cDNA Synthesis:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
Mutagenesis:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
SYBR Green Assay:Article Title: OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT Santa Cruz Cat# SC-8312; RRID: AB_671714 Anti-pAKT (Thr 308) Cell Signaling Technology Cat# 13038; RRID: AB_2629447 anti-FBN1 Abcam Cat# ab53076; RRID: AB_869770 anti-FBN1 Abcam Cat# ab124334; RRID: AB_10971846 Anti-FLAG Sigma-Aldrich Cat# F1804; RRID: AB_262044 Anti-G6PC Abgent Cat# AP5224c; RRID: AB_10665588 Anti-GST Cell Signaling Technology Cat # 2622; RRID: AB_331670 Anti-PCK1 Abclonal Cat # A2036; RRID: AB_2764060 Anti-phospho-PKA substrate (RRXS/T) Cell Signaling Technology Cat# 9624; RRID: AB_331817 Anti-Rhodopsin GeneScript Cat # A01628; RRID: AB_2622165 Anti-TUBA1A Sigma-Aldrich Cat# T6199; RRID: AB_477583 Donkey anti-Mouse, Alexa Fluor 488 Thermo Fisher Cat# A-21202; RRID: AB_141607 Goat anti-Mouse IgG HRP Bio-Rad Cat# 1706516; RRID: AB_11125547 Goat anti-Rabbit IgG HRP Bio-Rad Cat# 1706515; RRID: AB_11125142 Bacterial and Virus Strains DH5a Thermo Fisher 18265017 BL21 Thermo Fisher C600003 FLAG-OLFR734-HIS baculovirus This paper N/A FLAG-OLFR734-HIS (R186D) baculovirus This paper N/A FLAG-OLFR734-HIS (E173A) baculovirus This paper N/A Rho-OR4M1 lentivirus This paper N/A Rho-OR51E2 lentivirus This paper N/A CRE-Luc lentivirus This paper N/A RSV-Luc lentivirus This paper N/A Olfr734 shRNA lentivirus This paper N/A Olfr78 shRNA lentivirus This paper N/A pLKO lentivirus This paper N/A GFP adenovirus Han et al., 2015 N/A Rho-OR4M1 adenovirus This paper N/A Rho-OR51E2 adenovirus This paper N/A Rho-OLFR734 adenovirus This paper N/A Rho-OLFR734 (E173A) adenovirus This paper N/A Rho-OLFR734 (R186D) adenovirus This paper N/A Olfr734 shRNA adenovirus This paper N/A Olfr78 shRNA adenovirus This paper N/A US shRNA adenovirus Han et al., 2015 N/A RSV-Luc adenovirus This paper N/A CRE-Luc adenovirus This paper N/A Control shRNA AAV8 This paper N/A Olfr78 shRNA AAV8 This paper N/A Biological Samples Human cDNA Array OriGene CSRT101, CSRT302, HMRT104, HBRT301 cDNA, human adult normal tissue BioChain C1234063-10 (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Frozen human tissue slides Alenabio FDA903a-1, FDA903a-2, HuFTS246, HuFTS346 Frozen tissue section, human adult normal tissue BioChain T1234063 Chemicals, Peptides, and Recombinant Proteins Bluo-Gal INALCO 1758-0750 Carbenicillin, Disodium Salt INALCO 1758–9317 Cellfectin II Reagent Thermo Fisher 10362100 Collagenase Type IV Gibco 17104-019 Dexamethasone Sigma-Aldrich D4902 n-Dodecyl-b-D-Maltoside (DDM) Anatrace D310S Forskolin Sigma-Aldrich F3917 Gentamycin Sulfate INALCO 1758–1820 Glucagon Sigma-Aldrich G3157 Glucose Sigma-Aldrich G8270 [6, 6-2H] glucose Sigma-Aldrich 282650 GTPgS Sigma-Aldrich G8634 BODIPY-FL-GTPgS Thermo Fisher G22813 L-Glutathione reduced Sigma-Aldrich G4251 Insulin Novo Nordisk H20160213 IBMX Sigma-Aldrich I5879 IPTG Sigma-Aldrich I5502 Kanamycin Sulfate INALCO 1758–9316 Polyethylenimine Polysciences 23966–2 Proteinase K QIAGEN 19133 Sodium propionate Sigma-Aldrich P5436 Sodium pyruvate Sigma-Aldrich P2256 Tetracycline HCl INALCO 1758–9302 23SYBR Green qPCR Master Mix BiMake B21202 Protease inhibitor cocktail BiMake B14002 Critical Commercial Assays Alkaline Phosphatase Assay Kit Beyotime P0321 Alexa Fluor 647 Microscale Protein Labeling Kit Thermo Fisher A30009 Amplex Red Glucose/Glucose Oxidase Assay Kit Thermo Fisher A22189 BCIP/NBT Alkaline Phosphatase Color Development Beyotime C3206 cAMP ELISA Kit Cayman 581001 Cholesterol Fluorometric Assay Kit Cayman 10007640 Dual-Luciferase Reporter Assay System Promega E1980 Glucagon Quantikine ELISA Kit R&D systems DGCG0 LabAssay NEFA Wako 294–636 Mouse Insulin ELISA Kit Mercodia 10-1247-01 Mouse Leptin ELISA Kit RayBiotech ELM-Leptin-1 RevertAid First Strand cDNA Synthesis kit Thermo Fisher K1622 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies 200523 Serum Triglyceride Determination Kit Sigma-Aldrich TR0100 SYBR Green qPCR Master Mix Bimake B21203 Experimental Models: Cell Lines 3T3-L1 ATCC CL-173 HEK293T ATCC CRL-3216 HepG2 ATCC HB-8065 (Continued on next page) e2 Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hi-5 cells Thermo Fisher B85502 Sf9 ATCC CRL-1711 Experimental Models: Organisms/Strains C57BL/6J mouse Jackson Labs 664 Olfr734-/- mouse This paper N/A Gcgr-/- mouse This paper N/A Oligonucleotides Olfr734 sgRNA1: GAGAATAAATTCGGTCACGGTGG This paper N/A Olfr734 sgRNA2: ATGCTACCATCATGAACCGCCGG This paper N/A Olfr734 genotyping F: ACCTGCAAATGATACCACCGT This paper N/A Olfr734 genotyping R: TCCGGACAACCTGAGTGATG This paper N/A Gcgr sgRNA1: TGTTGGTGCTGTCATGTCTGG This paper N/A Gcgr sgRNA2: GGCTTAGGTTGTGGTGGCATTGG This paper N/A Gcgr genotyping F: CTTGGAGGCTTGGTTTGG This paper N/A Gcgr genotyping R: CTTTAGGGAACCACCACCTCT This paper N/A OR4M1 shRNA: AAAGTGGTGTCTGTGTTTCAT This paper N/A Olfr734 shRNA: GCAGGGCTATATCCACTTGTT This paper N/A Olfr78 shRNA: AGATTACTTTTGATGCCTGTCT This paper N/A qPCR Actin F: GTCCACCCCGGGGAAGGTGA Han et al., 2015 N/A qPCR Actin R: AGGCCTCAGACCTGGGCCATT Han et al., 2015 N/A qPCR G6pc F: GTGAATTACCAAGACTCCCAGGACTG This paper N/A qPCR G6pc R: GATGGAACCAGATGGGAAAGAGGAC This paper N/A qPCR Gcgr F: CAGCAGGTGATGTACACCGT This paper N/A qPCR Gcgr R: AGCAGCCAATCGATGACCAA This paper N/A qPCR Olfr734 F: CTCAGACTCGGGAAGTGCAG This paper N/A qPCR Olfr734 R: AAGCATCTTAGGCGCTGTGA This paper N/A qPCR Olfr78 F: ACGCTGCTGTCCTCAACAAT This paper N/A qPCR Olfr78 R: TGGGAGAGCACATTGGAGTG This paper N/A qPCR OR4M1 F: CCATCCATCTACATTTATGC This paper N/A qPCR OR4M1 R: ACTTCCTTGTTTCTCAATGT This paper N/A qPCR OR51E2 F: TCTACCTGCTGCTGCCTCCT This paper N/A qPCR OR51E2 R: GTCCTTGTCACAGCTGATCT This paper N/A qPCR Pck1 F: ATGTGGCCAGGATCGAAAGCAAGAC This paper N/A qPCR Pck1 R: CTTTCATGCACCCTGGGAACCTGG This paper N/A qPCR Pparg F: TGCACTGCCTATGAGCATT This paper N/A qPCR Pparg R: TGCGAGTGGTCTTCCATCAC This paper N/A Recombinant DNA pGEX-4T1 GE Healthcare 28954549 pGEX-Asprosin This paper N/A pCMV/SEAP Wang et al., 2014 N/A pCMV/SEAP-Asprosin This paper N/A pFastBac1-SF-OLFR734-H6 This paper N/A pFastBac1-SF-OLFR734 (R186D)-H6 This paper N/A pFastBac1-SF-OLFR734 (E173A)-H6 This paper N/A Bacmid-SF-OLFR734-H6 This paper N/A Bacmid-SF-OLFR734 (R186D)-H6 This paper N/A Bacmid-SF-OLFR734 (E173A)-H6 This paper N/A p3xFLAG-CMV-OLFR734 This paper N/A RSV-Luc Chen et al., 2017 N/A (Continued on next page) Cell Metabolism 30, 1–10.e1–e8, August 6, 2019 e3
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