L ring 3 5 3h tyrosine

Protein synthesis was measured as described by Gulve et al. [18 (link)] with the following modifications. L6 cells were plated in 48-well tissue culture plates and differentiated for 5 days. Cells were treated with 25 μM HMB for two hours in DMEM with 10% FBS and 0.8 mM L-Tyrosine and then for 1 h with 5 μM DEX in the absence or presence of effectors. Cells were then spiked with 1 μCi/ml of L-[ring-3,5–3H]-Tyrosine (Perkin Elmer, Waltham MA) and were incubated for 1 h. The reaction was stopped by placing the plates on ice. All wells were thoroughly washed twice with ice cold PBS media containing 2 mM non-radioactive L-Tyrosine (PBS-Tyr) and the cells then lysed in 0.1 ml of 0.1 mM NaOH/0.1% sodium deoxycholate. The cellular proteins were precipitated by adding 0.1 ml of cold 20% trichloroacetic acid (TCA; Sigma). This mixture was then incubated at 4°C for 15 min. After centrifugation (16,000×g for 10 min at 4°C), the pellet was washed once with cold 10% TCA and then, the precipitated proteins were dissolved in 0.1 ml of 1 M NaOH. An aliquot (5 μl) of the NaOH-solubilized material was used for total protein quantitation and the remaining dissolved proteins were neutralized with 1 M HCl, mixed with ReadySafe scintillation fluid (Beckman Coulter, Brea CA) and radiolabel determined with a scintillation counter (Beckman Coulter). Data was computed as dpm/μg of proteins.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum, and Penicillin-Streptomycin solution were purchased from Gibco-Thermo-Fisher Scientific (Grand Island, NY, USA). L-[2,3,4,5,6-3H] Phenylalanine and L-[Ring-3, 5-3H]-Tyrosine was purchased from Perkin-Elmer (Waltham, MA, USA), Prune extract-60% enriched polyphenol extract (PE60) was purchased from PL Thomas (Morristown, NJ, USA). All other chemicals were of reagent grade, and were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Protein synthesis was assesed by L-[ring-3,5-³H]-Tyrosine (Perkin Elmer, Waltham MA) as previously described by [19 (link)]. L-[ring-3,5-³H]-Tyrosine was added for 1 h to Neuro 2a cells that have preincubated for 2h in the absence (Control cells) or presence of 25 μM HMB (HMB cells) in DMEM with 10% FBS and 0.8 mM L-Tyrosine. Radioactivity in dissolved precipitates was counted using a scintillation counter (Beckman Coulter). Data was computed as dpm/μg of proteins.