KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti-UCP-1 (Western Blot) | Abcam | Cat# ab10983; RRID:AB_2241462 |
| Anti-UCP-1 (Immunohistochemistry) | Abcam | Cat# Ab23841; RRID:AB_2213764 |
| Anti-phospho HSL (Ser563) | Cell Signaling | Cat# 4139S; RRID:AB_2135495 |
| Anti-total HSL | Cell Signaling | Cat# 4107S; RRID:AB_2296900 |
| Anti-Tubulin | Cell Signaling | Cat# 3873S; RRID:AB_1904178 |
| Anti-phospho ERK | Cell Signaling | Cat# 9101L; RRID:AB_331646 |
| Anti-total ERK | Cell Signaling | Cat # 9102; RRID:AB_330744 |
| Anti-ATGL | Cell Signaling | Cat # 2439S; RRID:AB_2167953 |
| Phospho-p38 MAPK (Thr180/Tyr182) | Cell Signaling | Cat #9211; RRID:AB_331641 |
| Anti-p38 MAPK | Cell Signaling | Cat # 9212; RRID:AB_330713 |
| Anti-Beta Actin | Santa Cruz | Cat# sc-81178; RRID:AB_2223230 |
| Anti-Monoacylglycerol Lipase (MGL) | Abcam | Cat# ab180016 |
| IRDye 680RD goat anti–mouse IgG | Li-Cor | Cat# 925-68070; RRID:AB_2651128 |
| Dylight 800-conjugated goat anti–mouse IgG | Li-Cor | Cat# 925-32211; RRID:AB_2651127 |
| Anti-GAPDH | Santa Cruz | Cat# sc-47724; RRID:AB_627678 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Hoeschst 33342 | Invitrogen | H3570 |
| Seahorse XF Cell Mito Stress Test | Agilent | 103015-100 |
| Seahorse XFe24 FluxPak | Agilent | 100850-001 |
| iScript cDNA Synthesis Kit, 500 × 20 μl rxns | Biorad | 1708891BUN |
| 10x Tris/Glycine Buffer | Bio-Rad | 1610771EDU |
| 10x Tris/Glycine/SDS | Bio-Rad | 1610732EDU |
| 4–15% Mini-PROTEAN® TGX Precast Protein Gel | Bio-Rad | 4561086 |
| Cell Lysis Buffer (10X) | Cell Signaling | 9803 |
| RIPA Buffer (10X) | Cell Signaling | 9806 |
| 4-20% Mini-PROTEAN TGX gels | Bio-Rad | 456-1096 |
| Trans-Blot® Turbo Midi Nitrocellulose Transfer | Bio-Rad | 1704159 |
| 10x Tris/Glycine/SDS Buffer | Bio-Rad | 161-0772 |
| 10x Tris/Glycine Buffer | Bio-Rad | 161-0771 |
| Sybr Green | Applied Biosystems | 4385618 |
| Odyssey® Blocking Buffer (PBS) | Li-Cor | 927-40003 |
| MitoTracker® Red CMXRos | Life Technologies | M7512 |
| DMEM, high glucose, HEPES, no phenol red | Life Technologies | 21063-029 |
| Phusion® High-Fidelity PCR Kit | New England Biolabs, Inc | E0553L |
| Dual-Glo® Luciferase Assay System | Promega | E2940 |
| Glo Lysis Buffer, 1X | Promega | E2661 |
| QIAzol Lysis Reagent | QIAGEN | 7906 |
| RNeasy Mini Kit (250) | QIAGEN | 79306 |
| Paraformaldehyde solution 4% in PBS | Santa Cruz Biotechnology | CAS 30525-89-4 |
| (−)-Norepinephrine | Sigma | A7257 |
| 3,3′,5-Triiodo-L-thyronine sodium salt | Sigma | T6397 |
| 3-Isobutyl-1-methylxanthine | Sigma | I5879 |
| CL 316243, | Sigma | C5976 |
| Collagenase from Clostridium histolyticum | Sigma | C6885-1G |
| Dexamethasone | Sigma | D4902 |
| Free Glycerol Reagent | Sigma | F6428 |
| Glucose | Sigma | G8270 |
| Glycerol Standard | Sigma | G1394 |
| H-89 dihydrochloride hydrate | Sigma | B1427-5MG |
| Insulin from bovine pancreas | Sigma | I6634 |
| Isoproterenol | Sigma | I5627 |
| Rosiglitazone | Sigma | R2408 |
| DMEM | ThermoFisher Scientific | 11995065 |
| Lipofectamine® 3000 Transfection Reagent | ThermoFisher Scientific | L3000001 |
| Optimem | ThermoFisher Scientific | 31985047 |
| Bovine Serum Albumin - Fatty Acid Free | Roche | 3117057001 |
| FBS | Corning | 35-010-CV |
| Glucose strips - FreeStyle Lite Blood Glucose Test Strips | Abbott | 70819-70 |
| D-Glucose | Alfa Aesar | A16828 |
| Magnesium Chloride | Sigma | M8266 |
| Sodium Chloride | Fisher Scientific | BP318-212 |
| Potassium Chloride | Fisher Scientific | P217-500 |
| Sodium Phosphate Dibasic | Sigma | S3264 |
| LipidTox | ThermoFisher Reagent | H34476 |
| BODIPY | ThermoFisher Reagent | D3922 |
| DAPI | Life Technologies | D1306 |
| Sodium Phosphate Monobasic | Sigma | S8282 |
| Sodium Bicarbonate | Fisher Scientific | S3233-500 |
| Insulin (Human) - Humulin R | Lilly | 0002-8215-01 |
| CML-AGE | Synthesized in Laboratory per Kislinger et al., 1999 |
N/A |
| 2- [4- (acetylamino) phenyl] −4- Quinolinecarboxylic acid, methyl ester | Synthesized Per the Methods/Source outlined in Manigrasso et al., 2016 | N/A |
| Critical Commercial Assays | ||
| 2-CAT (A-N) Research ELISA | Rocky Mountain Diagnostics | BA E-5400 |
| cAMP Direct Immunoassay Kit | Abcam | Ab65355 |
| Glycerol/Triglyceride Determination Kit | Sigma | TR0100-1KT |
| NEFA HR(2) color reagent A | Wako Diagnostics | 99934691 |
| NEFA-HR(2) color reagent B | Wako Diagnostics | 99134891 |
| NEFA HR(2) Solvent A | Wako Diagnostics | 99534791 |
| NEFA HR(2) Solvent B | Wako Diagnostics | 99335191 |
| cAMP Assay Kit | Abcam | ab65355 |
| Pierce BCA Protein Assay Kit | ThermoFisher | 23225 |
| PKA Kinase Activity Assay Kit | Abcam | ab139435 |
| Experimental Models: Cell Lines | ||
| C3H/10T1/2, Clone 8 (ATCC® CCL-226) | ATCC American Type Culture Collection | ATCC CCL-226 |
| Experimental Models: Organisms/Strains | ||
| C57BL/6J mice | Jackson Laboratory | JAX:000664 |
| Ager Flox/ Flox mice | N/A | N/A |
| Adipoq Cre Recombinase mice | Jackson Laboratory | 28020 |
| Ager null mice | N/A | N/A |
| Recombinant DNA | ||
| pGL3- UCP1 | Koza Laboratory | N/A |
| PGL3 Basic | Promega | E1741 |
| Software and Algorithms | ||
| Graphpad Prism 8.02 | GraphPad Software | https://www.graphpad.com |
| Leica Digital Image Hub | Leica Biosystems | https://www.leicabiosystems.com |
| ImageJ | NIH | https://imagej.nih.gov |
| Fiji (distribution of ImageJ) | Schindelin et al., 2012 | https://fiji.sc |
| WAVE Seahorse XFe24 Analyzer | Agilent | https://www.agilent.com |
| LI-COR Odyssey Classic | Li-Cor | https://www.licor.com |
| Trans-Blot Turbo Transfer System | Bio-Rad | http://www.bio-rad.com |
| Image Studio Lite | Li-Cor | https://www.licor.com |