3h leucine

Manufactured by American Radiolabeled Chemicals

[3H]-Leucine is a radioactively labeled amino acid containing the tritium (3H) isotope. It is commonly used in research applications to study protein synthesis, cell metabolism, and related biological processes.

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3 protocols using 3h leucine

Neonatal Rat Cardiomyocyte Isolation and Culture

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Dulbecco's modified Eagle's medium (DMEM), trypsin-ethylenediaminetetraacetic acid, phosphate-buffered saline (PBS), l-glutamine, penicillin/streptomycin, fluorescent probe DCFH2-DA, TRIzol reagent, cDNA synthesis kit, SYBR Green PCR Master Mix, fluorescein isothiocyanate (FITC)-conjugated antibody and DAPI nuclear stain were purchased from Life Technologies, Grand Island, NY. FBS, normal horse serum (HRS), DPI, NAC, PE, trichloroacetic acid, NaOH and monoclonal anti-α-actinin (Sarcomeric) (A7732) were purchased from SIGMA Chemical Co., St. Louis, MO. Neonatal rat NeoMyts Kit was obtained from Cellutron Life Technology, Baltimore, MD. 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay kit was obtained from Promega, Madison, WI. [³H]-Leucine and [³H]-thymidine were obtained from American Radiolabeled Chemicals. Scintillation cocktail was obtained from RPI, Mount Prospect, IL. Anti-phospho p44/42 MAP kinase antibody (9101) was obtained from Cell Signaling, Danvers, MA. Lactosylceramide (LacCer) (palmitoyl) and other GSL stocks were purchased from Matreya LLC, Pleasant Gap, PA. All other chemicals used in this study were of the highest grade available from commercial suppliers.
Stock solution of GSLs, DPI and NAC were prepared in dimethyl sulfoxide (DMSO) and stored at −20°C until use.

Mishra S, & Chatterjee S. (2014). Lactosylceramide promotes hypertrophy through ROS generation and activation of ERK1/2 in cardiomyocytes. Glycobiology, 24(6), 518-531.

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Muscle Fiber Type Analysis

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We obtained ursolic acid (BML-CT105) and tomatidine (BML-GR335) from Enzo Life Sciences. Puromycin (P8833) was obtained from Sigma-Aldrich. [³H]Leucine (120 Ci/mmol) was obtained from American Radiolabeled Chemicals. Anti-myosin heavy chain I (BA-F8), anti-myosin heavy chain IIa (SC-71), anti-myosin heavy chain IIb (BF-F3), and anti-myosin heavy chain IIx (6H1) mouse monoclonal antibodies were obtained from the University of Iowa Developmental Studies Hybridoma Bank. Alexa Fluor 488-conjugated anti-mouse IgG₁, Alexa Fluor 350-conjugated anti-mouse IgG_2b, and Alexa Fluor 555-conjugated anti-mouse IgM were obtained from Life Technologies. Anti-FLAG mouse monoclonal antibody (F1804) was obtained from Sigma-Aldrich. Anti-puromycin mouse monoclonal antibody (MABE343) was obtained from EMD Millipore. All other antibodies were obtained from Cell Signaling Technology; these included anti-S6K (catalog number 2308S), anti-phospho-S6K (Thr-389) (catalog number 9234S), anti-Akt (catalog number 4691S), anti-phospho-Akt (Ser-473) (catalog number 4060S), anti-eukaryotic translational initiation factor 4E-binding protein 1 (4E-BP1) (catalog number 9644S), anti-phospho-4E-BP1 (Thr-37/46) (catalog number 2855S), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (catalog number 7074S) and anti-mouse IgG (catalog number 7076S).

Ebert S.M., Dyle M.C., Bullard S.A., Dierdorff J.M., Murry D.J., Fox D.K., Bongers K.S., Lira V.A., Meyerholz D.K., Talley J.J, & Adams C.M. (2015). Identification and Small Molecule Inhibition of an Activating Transcription Factor 4 (ATF4)-dependent Pathway to Age-related Skeletal Muscle Weakness and Atrophy. The Journal of Biological Chemistry, 290(42), 25497-25511.

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Drosophila Protein Signaling Pathway Protocol

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Reagents were obtained from the following sources: HRP-labeled anti-rabbit secondary antibody and the antibodies against Drosophila Phospho-70 S6 Kinase (Thr398) (#9209), Akt (#9272), phosphor-ERK (#9101), ERK (#4695), Akt (#9272), MYC (#2278), and the FLAG (#2368) epitope from Cell Signaling Technology (CST); Anti-Green Fluorescent protein (GFP) antibody from Aves Labs (GFP-1020); 8D12 Anti-Repo antibody from Developmental Studies Hybridoma Bank (DSHB); Alexa 488-, 568-, and 647-conjugated secondary antibodies and Complete Protease Cocktail from Roche; Schneider’s medium and Inactivated Fetal Bovine Serum (IFS) from Invitrogen; amino-acid-free Schneider’s medium from US Biologicals; [³H]-leucine from American Radiolabeled Chemicals, Inc.; leucine from Sigma (L8912); rapamycin from LC Laboratories (#R-5000); and Reliance One-Step Multiplex RT-qPCR Supermix from BIO-RAD. Fresh apples (Gala) were from Star Market. The dS6K antibody was a gift from Mary Stewart (North Dakota State University) and the Drosophila Sestrin antibody one from Jun Hee Lee (University of Michigan).

Gu X., Jouandin P., Lalgudi P.V., Binari R., Valenstein M.L., Reid M.A., Allen A.E., Kamitaki N., Locasale J.W., Perrimon N, & Sabatini D.M. (2022). Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila. Nature, 608(7921), 209-216.

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