FIG 5.
Hyperammonemia effects on ribosomal proteins are mediated by GSK- and mTORC1-independent effects. (A) Luciferase activity measuring transcriptional activity of β-catenin using the Topflash reporter assay (normalized to Renilla) comparing untreated (UnT) myotubes and those treated for 24 h with 10 mM AmAc in the presence or absence of 5 mM LiCl or 2 μM BIO. Data are depicted as fold changes over Topflash activity. (B) Representative immunoblots for β-catenin phosphorylated at Ser33/37 in untreated C2C12 myotubes or those treated with 10 mM AmAc for 6 or 24 h. Proteasome activity was blocked with 10 μM MG132 for the last 1 h to prevent ubiquitin-mediated degradation of phosphorylated β-catenin. (C) Fold changes in mRNAs for RPL5, -23, and -32 in myotubes treated for 24 h with and without 10 mM AmAc and/or rapamycin (Rap) (1 μg/ml). (D) Representative immunoblots of phosphorylated P70S6 kinase in response to 10 mM ammonium acetate for 24 h or 1 μg/ml rapamycin. All cellular experiments were performed in at least 3 biological replicates, blots were normalized to β-actin, and data are expressed as means ± SD. **, P < 0.01; ***, P < 0.001 (versus untreated cells). The antibody against active β-catenin recognizes the unphosphorylated, stable form of β-catenin.