| Antibodies |
| Anti-BLVRB rabbit monoclonal |
Sino Biological |
Cat# 13151-R009 |
| Anti-HO2 mouse monoclonal |
Abnova |
Cat# H00003163- M01; RRID:AB_425487 |
| Anti-IRS1 rabbit polyclonal |
EMD Millipore |
Cat# 06–248, RRID:AB_2127890 |
| Anti-INSR-beta rabbit polyclonal |
Santa Cruz |
Cat# sc-711, RRID:AB_631835) |
| Anti-INSR-beta phospho-Y1135,1136,1150,1151
rabbit monoclonal |
Invitrogen |
Cat# MA5–15148 RRID:AB_10982593 |
| Anti-IRS1 phospho-Y608 rabbit polyclonal |
EMD Millipore |
Cat# 09–432, RRID:AB_1163457 |
| Anti-NOS1 rabbit monoclonal |
Cell Signaling |
Cat# 4231 RRID:AB_2152485 |
| Anti-NOS2 rabbit monoclonal |
Santa Cruz |
Cat# sc-8310 RRID: AB_2152867 |
| Anti-NOS3 rabbit monoclonal |
Cell Signaling |
Cat#:9572 RRID:AB_329863 |
| Anti-NOS3 phospho-S1177 rabbit polyclonal |
ThermoFisher |
Cat# PA5–97371, RRID:AB_2809173 |
| Anti-NOS1 phospho-S1417 rabbit polyclonal |
Abcam |
Cat#ab5583 RRID:AB_304964 |
| Anti-AKT(pan) rabbit monoclonal |
Cell Signaling |
Cat# 4691, RRID:AB_915783 |
| Anti-AKT phospho-S473 rabbit polyclonal |
Cell Signaling |
Cat# 9271, RRID:AB_329825 |
| Anti-p97 mouse monoclonal |
Fitzgerald Industries |
Cat# 10R-P104a, RRID:AB_1287614 |
| Anti-GAPDH mouse monoclonal |
Abcam |
Cat# ab181602, RRID:AB_2630358 |
| Anti-AS160 rabbit monoclonal |
ThermoFisher |
Cat# MA5–14840, RRID:AB_10979793 |
| Anti-AS160 phospho-T642 rabbit polyclonal |
ThermoFisher |
4Cat# 44–1071G, RRID:AB_2533564 |
| Anti-ACADVL |
Santa Cruz |
Cat#sc-271225 RRID:AB_10609094 |
| Anti-FLAG M2 |
Sigma-Aldrich |
Cat# F3165, RRID:AB_259529 |
| Anti-AKR1A1 mouse monoclonal |
Origene |
Cat#TA500740 RRID:AB_11128818 |
| Anti-AKR1A1 mouse monoclonal |
Santa Cruz |
Cat# sc-100500 RRID:AB_1118799 |
| Anti-Histone3 rabbit |
Cell Signaling |
Cat#9705 RRID:AB_331563 |
| Anti-cytochrome C mouse monoclonal |
Cell Signaling |
Cat#12963 RRID:AB_2637072 |
| Anti-c-myc mouse monoclonal |
R&D system |
Cat#MAB3696 |
| Anti-Flag mouse monoclonal |
Sigma-Aldrich |
Cat#F1804 RRID:AB_262044 |
| Anti-His-Tag |
Cell Signaling |
Cat#2365 RRID:AB_2115720 |
| Anti-c-myc agarose |
Sigma-Aldrich |
Cat# A7470, RRID:AB_10109522 |
| Anti-FLAG M2-agarose |
Sigma-Aldrich |
Cat# A2220, RRID:AB_10063035 |
| Bacterial and virus strains |
| BL21-CodonPlus E. coli |
Agilent |
Cat# 230245 |
| Biological samples |
|
|
| De-identified human skeletal muscle samples,
human visceral adipose samples and human subcutaneous adipose tissue
samples |
Tissue Resource Core at University Hospitals
Cleveland/CWRU |
https://cwru.corefacilities.org/service_center/show_external/4499?name=tissue-resource-core
|
| Bovine liver |
Rockland Cat# BV-T278 |
Rockland Cat# BV-T278 |
| Fetal bovine serum (FBS) |
Sigma-Aldrich Cat# F4135 |
Sigma-Aldrich Cat# F4135 |
| Chemicals, peptides, and
recombinant proteins |
| Coenzyme A-agarose powder |
Sigma-Aldrich |
Cat# C7013 |
| Amylose-resin |
BioLabs |
Cat# E8021S |
| Activated thiol-Sepharose 4B |
GE Healthcare |
Cat# 17–0640-01 |
| Thiopropyl-Sepharose 6B |
GE Healthcare |
Cat# 17–0420-01 |
| Acetyl-CoA-agarose |
This study |
N/A |
| Palmitoyl-CoA-agarose |
Sigma-Aldrich |
Cat# P5297 |
| Glutathione (reduced)-agarose |
Invitrogen |
Cat# G2879 |
| SNO-modified activated thiol-Sepharose 4B |
This study |
N/A |
| SNO-modified coenzyme A (GSNO)-agarose |
This study |
N/A |
| Anti-TMT resin |
ThermoFisher |
Cat# 90060 |
| Protein A/G-Agarose |
Thermo Scientific |
Cat# 20423 |
| Ni-NTA-agarose |
Thermo Scientific |
Cat# 88221 |
| 3X FLAG peptide |
Sigma-Aldrich |
Cat# F4799 |
| [3H]-2-deoxyglucose |
PerkinElmer |
Cat# NET549250UC |
| [14C]-mannitol |
PerkinElmer |
Cat# NEC852050UC |
| Insulin (human) |
Sigma-Aldrich |
Cat# I9278 |
| PolyJet transfection reagent |
SignaGen |
Cat# SL100688 |
| DMEM, high glucose |
Thermo Scientific |
Cat# 11965092 |
| Penecillin/streptomycin |
Thermo Scientific |
Cat# 15140122 |
| Puromycin |
Thermo Scientific |
Cat# A1113803 |
| Krebs-Henseleit buffer |
Thermo Scientific |
Cat# J67795-AP |
| Sodium pyruvate |
Thermo Scientific |
Cat# 11360070 |
| Mannitol |
Sigma-Aldrich |
Cat# M4125 |
| 2-deoxyglucose |
Sigma-Aldrich |
Cat# D8375 |
| DPTA (diethylenetriaminepentaacetic acid) |
Cayman Chemical |
Cat# 33307 |
| NaNO2 |
Fluka |
Cat# 71759 |
| Bovine serum albumin (BSA) |
Sigma-Aldrich |
Cat# A2153 |
| Neocuproine |
Sigma-Aldrich |
Cat# N1501 |
| Protease inhibitor mix |
Roche |
Cat# 04693132001 |
| Phenylmethylsulfonyl fluoride (PMSF) |
Sigma-Aldrich |
Cat# P7626 |
| Imidazole |
Sigma-Aldrich |
Cat# I3386 |
| 2-mercaptoethanol |
Sigma-Aldrich |
Cat# M6250 |
| S-methylmethanethiosulfonate (MMTS) |
Sigma-Aldrich |
Cat# M9386 |
| Sodium ascorbate |
Sigma-Aldrich |
Cat# 11140 |
| Critical commercial assays |
| QuikChange II Site-Directed Mutagenesis
Kit |
Agilent |
Cat# 200523 |
| Bicinchoninic acid (BCA) protein quantitation
kit |
ThermoFisher |
Cat# 23225 |
| Accu-Chek glucose test strip |
Roche |
Cat#07299702001 |
| Ultra Sensitive Mouse Insulin ELISA kit |
Crystal Chem |
Cat# 90080 |
| Bilirubin assay kit |
Sigma-Aldrich |
Cat# MAK126 |
| iTRAQ Reagents Multiplex kit |
Sigma-Aldrich |
Cat# 4352135 |
| iodoTMTsixplex Isobaric Label Reagent Set |
ThermoFisher |
Cat# 90101 |
| Hemin Assay Kit |
BioVison |
Cat# K672 |
| InsR Kinase Enzyme System |
Promega |
Cat#V3901 |
| ADP-Glo™ Kinase Assay |
Promega |
Cat#V9101 |
| Deposited data |
| Raw images for Western blots |
This study |
doi:10.17632/zz3ktsyjds.1
|
| Experimental models: Cell
lines |
| HEK293 cells |
ATCC |
Cat# PTA-4488, RRID:CVCL_0045 |
| Rat L6 cells |
ATCC |
Cat# CRL-1458, RRID:CVCL_0385 |
| HEK293 SCAN-KO cells |
This study |
N/A |
| HEK293 SCAN-KO/SCAN-WT re-expressing
cells |
This study |
N/A |
| HEK293 SCAN-KO/SCAN-QTG/NAA-expressing
cells |
This study |
N/A |
| HEK293 SCAN-KO/SCAN-C109/188R-expressing
cells |
This study |
N/A |
| HEK293-SCoR-KO cells |
Stomberski Et al.9
|
N/A |
| L6 SCAN-KO cells |
This study |
N/A |
| L6 SCAN-KO/SCAN-WT re-expressing cells |
This study |
N/A |
| L6 SCAN-KO/SCAN-QTG/NAA-expressing cells |
This study |
N/A |
| L6 SACN-KO/SCAN-C109/188R-expressing
cells |
This study |
N/A |
| L6 Insr-KO cells |
This study |
N/A |
| L6 Insr-KO/INSR-WT re-expressing cells |
This study |
N/A |
| L6 Insr-KO/INSR-C1083S-expressing cells |
This study |
N/A |
| Experimental models:
Organisms/strains |
| Blvrbtm1(KOMP)Wtsi (SCAN-KO)
mice |
Model Animal Research Center, Nanjing
University |
RRID:MGI:5756156 |
| B6.Cg-Lepob/J (ob/ob) mice |
Jax.org
|
Cat# 000632, RRID:IMSR JAX:00 0632 |
| Oligonucleotides |
| SCAN-KO genotyping primer Blvrb-FRT-tF1
5’-AGAGTTTGGGTCCTCCCTTTCCT-3’ |
ThermoFisher |
N/A |
| SCAN-KO genotyping primer Common-En2-R
5’-CCAACTGACCTTGGGCAAGAACAT-3’ |
ThermoFisher |
N/A |
| rat sgRNA 1 (Blvrb/SCAN)
5’-CCCCTCTGACGGTAACCTGC-3’ |
GenScript |
N/A |
| rat sgRNA 2 (Blvrb/SCAN)
5’-TCGGTGCCACCGGAAGGACC-3’ |
GenScript |
N/A |
| rat sgRNA 1 (Insr)
5’-TATCGACTGGTCCCGCATCCTGG-3’ |
GenScript |
N/A |
| rat sgRNA 2 (Insr)
5’-GCCTGATTATCAACATCCGAGGG-3’ |
GenScript |
N/A |
| Mutagenesis primers (See Table S5) |
GenScript |
See Table S5
|
| Recombinant DNA |
| pcDNA3 human IRS1-FLAG |
GenScript |
Cat# OHu24973 |
| pcDNA3 human INSR-FLAG |
Genscript |
Cat# OHu24951 |
| pcDNA3.1 vector |
ThermoFisher |
Cat# V79020 |
| pcDNA3.1 human HO2 |
This study |
N/A |
| pcDNA3.1 human HO2 C127R mutant |
This study |
N/A |
| pcDNA3.1 human HO2 C265R mutant |
This study |
N/A |
| pcDNA3.1 human HO2 C282R mutant |
This study |
N/A |
| pcDNA3.1 human HO2 1–296 |
This study |
N/A |
| pcDNA3.1 human HO2 65–316 |
This study |
N/A |
| pcDNA3.1 human HO2 130–316 |
This study |
N/A |
| pcDNA3.1 human HO2 195–316 |
This study |
N/A |
| pcDNA3.1 human SCAN(BLVRB) |
This study |
N/A |
| pcDNA3.1 human SCAN QTG-NAA mutant |
This study |
N/A |
| pcDNA3.1 human SCAN C109R mutant |
This study |
N/A |
| pcDNA3.1 human SCAN C188R mutant |
This study |
N/A |
| pcDNA3.1 human SCAN C109/188R mutant |
This study |
N/A |
| pcDNA3 human INSR C825A mutant |
This study |
N/A |
| pcDNA3 human INSR C834A mutant |
This study |
N/A |
| pcDNA3 human INSR C1083A mutant |
This study |
N/A |
| pcDNA3 human NOS3 (eNOS) |
Ozawa Et al. 70
|
N/A |
| pET21b vector |
Sigma-Aldrich |
Cat# 69741 |
| pET21b human HO2–6xHis |
This study |
N/A |
| pET21b human
HO2–6xHis-195–316 |
This study |
N/A |
| pET21b human SCAN-6xHis |
This study |
N/A |
| pET21b human SCAN-6xHis QTG-NAA mutant |
This study |
N/A |
| pET21b human SCAN-6xHis C109/188R mutant |
This study |
N/A |
| Human BLVRB CRISPR/Cas9 KO plasmid |
Santa Cruz Biotechnology |
Cat# sc-405988 |
| Human BLVRB HDR plasmid |
Santa Cruz Biotechnology |
Cat# sc-405988- HDR |
| Rat eSpCas9-Blvrb targeting plasmid |
GenScript |
N/A |
| Rat eSpCas9-Insr targeting plasmid |
GenScript |
N/A |
| Software and algorithms |
| GraphPad Prism, v8 |
GraphPad Inc |
https://www.graphpad.com/
|
| Other |
| High-fat diet for mice (60 kcal% fat
diet) |
Research Diets, Inc. |
D12492 |
| Teklad rodent chow diet |
Envigo |
P3000 |
