All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Washington. PP2Cm germ-line knockout (KO) mice with C57BL/6 background were generated as previously described (Lu et al., 2009 (link)). To overexpress GLUT1, the PP2Cm-KO mice were crossed with cardiac-specific GLUT1-TG mice for more than 6 generations into a C57BL/6 background. Mice were maintained on standard chow diet and water available ad libitum in a vivarium with a 12-hour light/dark cycle at 22°C. BCAAs (weight ratio, isoleucine: leucine: valine =0.8:1.5:1, Sigma-Aldrich) were administrated by oral gavage for 7 days at 1.5 mg/g body weight/day (D’Antona et al., 2010 (link)). Compound BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid) were purchased from Santa Cruz biotechnology and administrated by oral gavage at 40 mg/kg body weight/day for 7 days as previously described (Sun et al., 2016 (link)). Blood samples for BCAA level measurement were collected via retro-orbital bleeding. Additional details are provided in the Supplemental Experimental Procedures .
Bcaas
Manufactured by Merck Group
Sourced in United States
BCAAs is a laboratory equipment product manufactured by Merck Group. It is designed for the measurement and quantification of branched-chain amino acids, which are essential components in various biological and biochemical analyses.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Merck Group (2 mentions)
Compound bt2 3 6 dichlorobenzo b thiophene 2 carboxylic acid, by Santa Cruz Biotechnology (1 mentions)
L valine, by Merck Group (1 mentions)
L isoleucine, by Merck Group (1 mentions)
L leucine, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
3 methyladenine 3 ma, by MedChemExpress (1 mentions)
Gabapentin, by Merck Group (1 mentions)
Chloroquine, by MedChemExpress (1 mentions)
Gabapentin, by MedChemExpress (1 mentions)
Rapamycin, by MedChemExpress (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Ab56023, by Abcam (1 mentions)
Ab140010, by Abcam (1 mentions)
5 protocols using bcaas
1
Metabolic Regulation via GLUT1 and PP2Cm Modulation
2
Vitamin D3 and BCAA Analysis
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1α,25-Dihydroxyvitamin D3 (BML-DM200) was purchased from Enzo Life Sciences and was used at a final concentration of 100 nM. BCAAs were purchased from Sigma Aldrich and used at the following concentrations: L-leucine 115 µM (Cat# L8000), L-valine 200 µM (Cat# V0513), and L-isoleucine 60 µM (Cat# I7403).
3
Modeling BCAA Dysregulation in hPSC-CMs
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Cor4.U control hPSC-CMs were purchased from Ncardia (Leiden, The Netherlands) and were plated on gelatin (0.1% diluted in PBS) coated coverslips at a density of 5000 cells per coverslip following manufacturer’s instructions. The generation and use of these cells were conform to the declaration of Helsinki. Cells were first plated in complete culture medium (Ncardia) for 24 h and then cultured for 3–4 days in PMC medium (Ncardia) with modified branched chain amino acid (BCAA) concentration. BCAA concentrations in control condition were normalized to 800 nmol/L and supplemented to similar concentrations as quantified by NMR in plasma samples from Bcat2p.Q300*/p.Q300* mice (i.e. valine: 10 mmol/L; leucine: 7.5 mmol/L; isoleucine: 4.5 mmol/L). BCAAs were purchased from Sigma-Aldrich and diluted in PMC medium. Rapamycin (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mmol/L. For experiments, Rapamycin was used at a concentration of 500 nmol/L diluted in PMC medium (final DMSO concentration 0.0025%).
4
Evaluating Small Molecule Interactions
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The following small molecules were used: cisplatin and Leu (Sigma-Aldrich), chloroquine (CQ), rapamycin, gabapentin, and 3-methyladenine (3-MA) (MedChemExpress, Princeton, NJ, USA), Ac-Leu-NH2 (BACHEM, Switzerland), BCAAs (Anhui BBCA Pharmaceuticals Co., Ltd., China). The cisplatin, CQ, gabapentin, 3-MA, Leu, Ac-Leu-NH2, and BCAAs were dissolved in phosphate buffer saline, and rapamycin was dissolved in dimethyl sulfoxide (Sigma-Aldrich).
5
BMP-7 and Gremlin1 Pathway Analysis
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Trizol reagent, the reverse transcription (RT) kit, and semiquantitative real-time PCR kit were purchased from Takara (Takara, Japan). Rabbit polyclonal antibodies specific for BMP-7 (ab56023, Abcam, USA) and gremlin1 (ab140010, Abcam, USA), Smad1/5/8 (sc-7965, Santa Cruz, Canada), phosphorylated (p)-SMAD1/5/8 (SMAD1, 5-phospho-Ser463/465, and SMAD8 phospho-Ser 426/428 antibodies; 13820, Cell Signaling Technology, Canada), and p-Smad2/3 (YP0362, Immunoway Biotechnology, China) were used in this study. Secondary antibodies were obtained from Jackson (Jackson, USA). The TGF-β1 and FN enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boster (EK0515, Boster, Wuhan, China). Low-glucose Dulbecco’s modified Eagle medium (DMEM) was purchased from Hyclone (Hyclone, USA). Penicillin/streptomycin and trypsin were obtained from North China Pharmaceutical (NCPC, China). BCAAs were purchased from Sigma-Aldrich Company (USA). Glucose was purchased from China Otsuka Pharmaceutical Company (Otsuka, China). Mannitol was purchased from Shandong JinYang Pharmaceutical (Shandong JinYang Pharmaceutical, China).
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