Fig 1: Met activates ERK1/2 and mTORC1 through the T1R1/T1R3 receptor. Serum-starved C2C12 myoblasts in KRBH were treated with 1 mM/L IMP for 15 min prior to treatment with 50 mM Met for 60 min. p-S6K1 and p-mTOR protein levels were analyzed by Western blotting, and ß-actin was used as a loading control (A); C2C12 myoblasts were treated as in (A), loaded with Fluo8-AM, and pretreated with disodium 5'-inosinate (IMP) for 30 min prior to Met stimulation. Data are the mean ± SEM of the relative fluorescence units (RFU) values of three independent experiments (B); The control (Con) esiRNA and T1R1 esiRNA (Sigma, St. Louis, MO, USA) were transfected into C2C12 myoblasts in KRBH were stimulated with 50 mM Met for 60 min. T1R1, p-ERK1/2, p-S6K1, and p-mTOR protein levels were analyzed by Western blotting, and ß-actin was used as a loading control (C). C2C12 myoblasts were treated as in (C) and loaded with Fluo8-AM; Data are the mean ± SEM of the RFU values of three independent experiments (D). “+” means to add, “-” means not to add. Results were represented as mean ± SD, * p < 0.05, ** p < 0.01.
Fig 2: T1R1/T1R3-mediated amino acids regulate ERK1/2 and mTORC1 through stimulation of Ca2+ in C2C12 myotubes. RNA was isolated from C2C12 myoblasts of different differentiation times for quantitative polymerase chain reaction (Q-PCR) analysis (A); Serum-starved C2C12 cells in Krebs-Ringer’s solution (KRBH) were treated with the indicated concentrations of BAPTA-AM 15 min prior to treatment with AA for 60 min, p-S6K1, p-mTOR protein levels were analyzed by Western blotting, and ß-actin was used as a loading control (B); C2C12 myoblasts in KRBH were treated with the indicated concentrations of U0126 15 min prior to treatment with AA for 60 min. Immunoblots were performed with the indicated antibodies (C); C2C12 cells in KRBH were treated with the 1 mM/L IMP or 10 µM/L BAPTA-AM 15 min prior to treatment with AA for 60 min. Immunoblots were performed with the indicated antibodies (D). “+” means to add, “-” means not to add.
Supplier Page from Abcam for Anti-TR1 antibody