KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonal anti-Ism1 | Abcam | Cat#AB103338; RRID:AB_10710279 |
| Rabbit monoclonal anti-p-AKT (Ser473) | Cell Signaling Technology | Cat#4060; RRID:AB_2315049 |
| Rabbit monoclonal anti-p-AKT (Thr308) | Cell Signaling Technology | Cat#13038; RRID:AB_2629447 |
| Rabbit monoclonal anti-Akt (pan) (C67E7) | Cell Signaling Technology | Cat#4691; RRID:AB_915783 |
| Rabbit polyclonal anti-p-ERK1/2 (Thr202/Tyr204) | Cell Signaling Technology | Cat#9101; RRID:AB_331646 |
| Rabbit polyclonal anti-ERK1/2 | Cell Signaling Technology | Cat#9102; RRID:AB_330744 |
| Rabbit polyclonal anti-p-S6 Ribosomal Protein (Ser235/236) | Cell Signaling Technology | Cat#2211; RRID:AB_331679 |
| Rabbit monoclonal anti-p-PRAS40 (Thr246) (C77D7) | Cell Signaling Technology | Cat#2997; RRID:AB_2258110 |
| Rabbit polyclonal anti-p-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) | Cell Signaling Technology | Cat#3021; RRID:AB_331578 |
| Rabbit polyclonal anti-p-IGF-I Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) (19H7) | Cell Signaling Technology | Cat#3024; RRID:AB_331253 |
| Rabbit monoclonal anti-IGF-I Receptor β (D23H3) | Cell Signaling Technology | Cat#9750; RRID:AB_10950969 |
| Phospho-Tyrosine (P-Tyr-1000) MultiMab Rabbit mAb mix | Cell Signaling Technology | Cat#8954 RRID:AB_2687925 |
| Biotin conjugated antibody anti-Ism1 6E1B6 | This paper | N/A |
| Biotin conjugated antibody anti-Ism1 7BF11 | This paper | N/A |
| Rabbit polyclonal anti-p-FoxO1 (Ser256) | Cell Signaling Technology | Cat#9461; RRID:AB_329831 |
| Rabbit monoclonal anti-FoxO1 (C29H4) | Cell Signaling Technology | Cat#2880; RRID:AB_2106495 |
| Rabbit polyclonal anti-p-GSK-3α/β (Ser21/9) | Cell Signaling Technology | Cat#9331; RRID:AB_329830 |
| Rabbit monoclonal anti- p-GSK-3β (Ser9) (D85E12) | Cell Signaling Technology | Cat#5558; RRID:AB_10013750 |
| Rabbit polyclonal anti-p-PDK1 (Ser241) | Cell Signaling Technology | Cat#3061; RRID:AB_2161919 |
| Rabbit monoclonal anti-PDGFRα (D1E1E) | Cell Signaling Technology | Cat# 3174, RRID:AB_2162345 |
| Rabbit monoclonal anti-p-mTOR (Ser2448) (D9C2) | Cell Signaling Technology | Cat#5536; RRID:AB_10691552 |
| Rabbit monoclonal anti-mTOR (7C10) | Cell Signaling Technology | Cat#2983; RRID:AB_2105622 |
| Rabbit monoclonal anti-p-PKA Substrate (RRXS*/T*) (100G7E) | Cell Signaling Technology | Cat#9624; RRID:AB_331817 |
| Rabbit monoclonal anti-PI3 Kinase Class III (D4E2) | Cell Signaling Technology | Cat#3358; RRID:AB_2299768 |
| Rabbit monoclonal anti-DYKDDDDK(FLAG) Tag (D6W5B) | Cell Signaling Technology | Cat#14793; RRID:AB_2572291 |
| Rabbit polyclonal anti-Acetyl-CoA Carboxylase | Cell Signaling Technology | Cat#3662; RRID:AB_2219400 |
| Rabbit monoclonal anti-Fatty Acid Synthase (C20G5) | Cell Signaling Technology | Cat#3180; RRID:AB_2100796 |
| Mouse monoclonal anti-SREBP1(2A4) | Thermo Fisher Scientific | Cat#MA5-16124; RRID:AB_11152118 |
| Mouse monoclonal anti- beta Actin [AC-15] (HRP) | Abcam | Cat#AB49900; RRID:AB_867494 |
| Mouse monoclonal anti-Glucose Transporter GLUT4 (1F8) | Cell Signaling Technology | Cat# 2213, RRID:AB_823508 |
| Rabbit polyclonal anti-Glucose Transporter GLUT4 | Abcam | Cat#AB654; RRID:AB_305554 |
| Sheep anti-mouse IgG (HRP) | Cytiva (GE) | CAT#NA931; RRID:AB_772210 |
| Donkey anti-rabbit IgG (HRP) | Cytiva (GE) | CAT#NA934; RRID:AB_772206 |
| Bacterial and Virus Strains | ||
| One Shot™ TOP10 Chemically Competent E. coli | Thermo Fisher Scientific | Cat#C404010 |
| AAV8-Ism1-FLAG | Vector Biolabs | N/A |
| AAV8-GFP | Vector Biolabs | Cat#7061 |
| Biological Samples | ||
| Adult subcutaneous adipose tissues | Beth Israel Deaconess Medical Center | IRB 2011P000079 |
| Human plasma samples | (Crimarco et al., 2020) | NCT03718988 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Mouse recombinant Ism1-his protein | This paper | N/A |
| Human recombinant insulin | Millipore Sigma | Cat#91077C |
| Human recombinant PDGF-BB | R&D Systems | Cat#220-BB |
| Bovine Serum Albumin | Millipore Sigma | Cat#A7906 CAS:9048-46-8 |
| Deoxy-D-glucose, 2-[1,2-3H (N)]-, Aqueous Sol, Specific Activity: 5-10 Ci (185-370 GBq)/mmol | Perkin-Elmer | Cat#NET328A001MC |
| Acetic acid, sodium salt, [3 H]- 5 mCi (185 MBq) | Vitrax | Cat#VT211 |
| L-[3,4,5-3H(N)]-Leucine, Specific Activity: 100 to 150 Ci/mmol (3.7 to 5.56 TBq/mmol), 250μCi (9.25MBq) | Perkin-Elmer | Cat#NET460250UC |
| Rapamycin | Cell Signaling Technology | Cat#9904 |
| LY294002 | Cell Signaling Technology | Cat#9901 |
| Wortmannin | Cell Signaling Technology | Cat#9951 |
| Torin1 | Selleckchem | Cat#S2827 |
| PIK-75 HCl | Selleckchem | Cat#S1205 |
| Omipalisib | Selleckchem | Cat#S2658 |
| Sapanisertib (INK-128) | Selleckchem | Cat#S2811 |
| Linsitinib (OSI-906) | Selleckchem | Cat#S1091 |
| LDC1267 | Selleckchem | Cat#S7638 |
| S6K1 Inhibitor II, DG2 | Millipore Sigma | Cat#559274 |
| Hoechst 33342 | Thermo Fisher Scientific | Cat#H3570 |
| Oil Red O solution | Millipore Sigma | Cat#O1391 |
| 2x SYBR Green qPCR master mix | Bimake | Cat# B21203 |
| Trizol | ThermoFisher | Cat# 15-596-026 |
| High-capacity cDNA Reverse Transcription kit | Biosystems | Cat# 4368814 |
| DMEM/F12 (1:1) | ThermoFisher | Cat# 11320082 |
| DMEM/F12 + Glutamax | ThermoFisher | Cat# 10565-042 |
| Expi293 Expression medium | ThermoFisher | Cat# A1435101 |
| Opti-MEM reduced serum media | ThermoFisher | Cat# 31985062 |
| HBSS buffer | Gibco | Cat# 14175-095 |
| Potassium chloride | Quality Biological | Cat# 112-033-101 |
| Sodium bicarbonate | Sigma | Cat# S6297 |
| UltraPure™ 0.5M EDTA, pH8.0 | Invitrogen | Cat# 15575-038 |
| Williams E media | Quality Biological | Cat# 112-033-101 |
| Corning™ Regular Fetal Bovine Serum | Corning | Cat# 35-010-CV |
| DMEM high glucose | Sigma | Cat# D6429 |
| HEPES buffered saline | Sigma-Aldrich | Cat# 51558 |
| Sodium pyruvate | ThermoFisher | Cat# 11360070 |
| Dexamethasone | Sigma | Cat# D1756 |
| Biotin | Sigma | Cat# B-4639 |
| D-Pantothenic acid | Sigma | Cat# P5155 |
| Human transferrin | Sigma | Cat# T-2252 |
| Cortisol | Sigma | Cat# H-0888 |
| Triiodothyronine | Sigma | Cat# T-6397 |
| IBMX | Sigma | Cat# I-5879 |
| Rosiglitazone | Cayman | Cat# 71740 |
| Calcium chloride | Sigma | Cat#100-43-52-4 |
| Dispase II | Roche | Cat# 04942078001 |
| D-(+)-Glucose | Sigma | Cat# 50-99-7 |
| Primocin | FisherScientific | Cat# NC9141851 |
| Trypsin/EDTA 0.25% | Gibco | Cat# 25200-056 |
| Penicillin/Streptomycin | Gibco | Cat# 15140-122 |
| Collagenase IV | Sigma | Cat# C5138 |
| Collagenase D | Roche | Cat# 11088882001 |
| PBS | Gibco | Cat# 10010-023 |
| 10x PBS | Gibco | Cat# 70011044 |
| Percoll | Sigma | Cat# P1644 |
| Ethanol | FisherScientific | Cat# 22-032-601 |
| Glutamax | ThermoFisher | Cat# 35050061 |
| Trypan Blue Stain (0.4%) | Invitrogen | Cat# T10282 |
| Isopropanol | FisherChemical | Cat# BP2632-4 |
| Formalin | SIP Brand | Cat# C4320 |
| Rat tail Collagen I | Corning | Cat# 354236 |
| Metformin | Sigma | Cat# 317240 |
| GW4064 | Sigma | Cat# G5172 |
| Imidazole | Sigma | Cat# I5513 |
| Hematoxylin Solution, Mayer’s | Millipore Sigma | Cat#MHS1 |
| Immobilon Crescendo Western HRP substrate | Millipore Sigma | Cat t# WBLUR0500 |
| SuperSignal West Femto HRP substrate | ThermoScientific | Cat# 34095 |
| SeeBlue Plus2 prestained standard | Invitrogen | Cat# LC5925 |
| RIPA buffer (10x) | Cell Signaling | Cat# 9806S |
| NuPAGE LDS sample buffer (4x) | Invitrogen | Cat# NP0007 |
| 2-mercaptoethanol | FisherChemical | Cat# O3446I |
| PhosSTOP | Roche | Cat# 04906837001 |
| cOmplete Tablets | Roche | Cat# 04693124001 |
| Critical Commercial Assays | ||
| Proteome Profiler Human Phospho-Kinase Array Kit | R&D Systems | Cat#ARY003C |
| AlphaLISA SureFire Ultra AKT 1/2/3 (pS473) Assay Kit - High Volume | Perkin-Elmer | Cat#ALSU-PAKT-B-HV |
| Ultra-Sensitive Mouse Insulin ELISA kit | Crystal Chem Inc | Cat#90080; RRID:AB_2783626 |
| Infinity triglyceride measurement kit | Thermo Fisher Scientific | Cat#TR22421 |
| Triglyceride colorimetric assay kit | Cayman | Cat#10010303 |
| Deglycosylation Mix II | New England BioLabs | Cat#P6044 |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | Cat#23225 |
| Glycerol release assay | Abcam | Cat#ab133130 |
| Plasma Membrane Protein Extraction Kit | Abcam | Cat#ab65400 |
| Deposited Data | ||
| Raw and processed proteomics data | ProteomeXchange Consortium via Proteomics Identification (PRIDE) | PXD026921 JPost: JPST001229 |
| Experimental Models: Cell Lines | ||
| 3T3-F442A cells | Millipore Sigma | Cat#00070654; RRID:CVCL_0122 |
| AML12 cells | ATCC | Cat#CRL-2254; RRID:CVCL_0140 |
| SGBS cells | (Wabitsch et al., 2001) | RRID:CVCL_GS28 |
| Primary human skeletal muscle cells | Cook Myocytes | Cat#SK-1111 |
| 3T3-L1 cells | ATCC | Cat#CL-173; RRID:CVCL_0123 |
| Expi293F Cells | Thermo Fisher Scientific | Cat# A14527 |
| Primary mouse white and brown adipocytes | This paper | N/A |
| Experimental Models: Organisms/Strains | ||
| Mouse: C57BL/6J | Jackson Laboratory | Cat#000664; RRID:IMSR_JAX:000664 |
| Mouse: Ism1 whole-body knockout | This paper | N/A |
| Oligonucleotides | ||
| shRNA target sequencing against Ism1 (shRNA1 Ism1): 5’-CACCGGTCACCATAGAGGTGGTTGACGAATCAACCACCTCTATGGTGACC-3’ | This paper | N/A |
| shRNA target sequencing against Ism1 (shRNA2 Ism1): 5’-CACCGCGGGAAGTGAGGAGTTTAATCGAAATTAAACTCCTCACTTCCCGC-3’ | This paper | N/A |
| Genotyping primers: Ism1 KO: 5′ -CTATGCTATGCCCAGTGTCTCTCTCTG- 3′; 5′- CAAACTGACCAGAGTCCCTCCTTCAA- 3′, Ism1 WT: 5′- GAACACTGAGGAAGTTGCTGTCA -3′; 5′- ATGGCCCTGACTCCGAAGCAGAA -3. | This paper | N/A |
| Primers for q-RT-PCR, see Table S4 | This paper | N/A |
| Recombinant DNA | ||
| Ism1-his expression plasmid | This paper | N/A |
| BLOCK-iT™ U6 RNAi Entry Vector | Thermo Fisher Scientific | Cat#K494500 |
| pAd/BLOCK-iT™-DEST RNAi Gateway® Vector | Thermo Fisher Scientific | Cat#K494100 |
| Software and Algorithms | ||
| ImageJ | Schneider et al., 2012 | https://imagej.nih.gov/ij/; RRID:SCR_003070 |
| GraphPad Prism version 8.0 | GraphPad Software, San Diego, California USA | http://www.graphpad.com/; RRID:SCR_002798 |
| Adobe Illustrator | Adobe systems | RRID: SCR_010279 |
| Other | ||
| NAFLD diet | ResearchDiet | Cat#D09100310 |
| HFD diet (60%) | ResearchDiet | Cat# D12492 |
| ExpiFectamine 293 Transfection Kit | Thermo Fisher Scientific | Cat#A14524 |
| Corning® 125 mL Polycarbonate Erlenmeyer Flask with Vent Cap | Corning | Cat# 431143 |
| His GraviTrap Talon columns | Millipore Sigma | Cat#GE29-0005-94 |
| HisProbe-HRP | Thermo Scientific | Cat#15165 |
| Slide-A-Lyzer Dialysis Cassette 3.5 MWCO | Thermo Fisher Scientific | Cat#66330 |
| Countess II FL Automated Cell Counter | Life Technologiy | Cat# AMQAF1000 |
| Blood Glucose meter | OneTouch UltraMini meter | N/A |
| Blood Glucose Strips | GenUltimate | Cat# 100-50 |
| 70um cell strainer | BD Falcon | Cat# 352350 |
| 25G x 7/8 Needle | BD | Cat# 305124 |
| Petri dish Fisherbrand™ | FisherScientific | Cat# FB0875713 |
| Fisherbrand™ Variable-Flow Peristaltic Pump | FisherScientific | Cat# 13-876-2 |
| Perfusion tubing connectors 3/32” Polypropylene Coupler | United States plastic Corp | Cat# 65600 |
| Perfusion tubing 1/32” ID x 3/32” OD Silicone | United States plastic Corp | Cat# 57286 |
| Immobilon-P PVDF Membrane | Merck | Cat# IPVH00010 |
| NuPAGE 4-12% Bis Tris gels | Invitrogen | Cat# WG1403BX10 |