FIG 4.

Skeletal muscle hyperammonemia does not alter GSK3β expression or activity. (A to C) Representative immunoblots of phosphorylated and total GSK3β from untreated (UnT) C2C12 myotubes or those treated with 10 mM AmAc for 6 or 24 h (A), skeletal muscle from portocaval anastomosis (PCA) and sham-operated pair-fed control (Sham) rats (n = 5 each) (B), and skeletal muscle of patients with cirrhosis (CIR) and controls (CTL) (n = 5 each) (C). (D) Representative photomicrographs and myotube diameters of differentiated C2C12 myotubes depleted of GSK3β or GSK3α or transfected with a vector with a random construct (shRan), treated with and without 10 mM ammonium acetate (AmAc). Scale bars, 40 μm. (E) Representative immunoblots of dephosphorylated active (A) and total (T) β-catenin, as well as GSK3β, in untreated C2C12 myotubes or those treated with 10 mM AmAc for 24 h in the presence or absence of GSK3β inhibitors, 5 mM lithium chloride (LiCl) or 2 μM BIO (6-bromoindirubin-3′-oxime), that activate β-catenin. Densitometry of blots from active β-catenin were normalized to β-actin. (F) Representative immunoblots of GSK3α and GSK3β and immunoblots and densitometry of RPL5, RPL23, RPL32, P70S6K, and β-actin in myotubes with silencing of GSK3α and GSK3β with and without 10 mM AmAc for 24 h. Expression of total P70S6K was used to determine the response of a nonhousekeeping, regulatable protein with hyperammonemia. 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.