Skip to main content
. 2019 Jul 29;39(16):e00451-18. doi: 10.1128/MCB.00451-18

FIG 3.

FIG 3

Reduced skeletal muscle β-catenin expression during hyperammonemia. (A to C) Representative immunoblotting and densitometry of cMYC and upstream binding factor 1 (UBF1) expression in untreated (UnT) murine C2C12 myotubes or those treated with 10 mM AmAc for 24 h (A); skeletal muscle from portocaval anastomosis (PCA) and sham-operated, pair-fed (Sham) control rats (n = 5 each) (B); and skeletal muscle of patients with cirrhosis (CIR) and controls (CTL) (n = 5 each) (C). (D to F) Representative immunoblotting and densitometry of total (T) and active, dephosphorylated (A) β-catenin expression from untreated C2C12 myotubes or those treated with 10 mM AmAc for 24 h (D); from the gastrocnemius muscle of PCA and sham-operated, pair-fed control rats (n = 5 each) (E); and from the skeletal muscle of patients with cirrhosis and control subjects (n = 5 each) (F). (G) Representative immunoblotting and densitometry of total and activated β-catenin as well as its transcriptional targets cMYC and UBF1 in untreated HEK cells or those treated with 10 mM ammonium acetate for 6 or 24 h. (H) Luciferase activity measuring transcriptional activity of β-catenin using Topflash reporter activity (normalized to Renilla) comparing untreated myotubes and those treated for 24 h with 10 mM AmAc, 5 mM lithium chloride (LiCl), or 2 μM BIO (6-bromoindirubin-3′-oxime) (small-molecule inhibitor of GSK3β with resultant activation of β-catenin), expressed as fold changes over untreated cells. (I) Representative photomicrographs of immunofluorescence of active, unphosphorylated β-catenin expression (green) in murine C2C12 myotubes treated with 10 mM AmAc or 2 μM BIO for 24 h. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 40 μm. **, P < 0.01; ***, P < 0.001 (versus untreated cells). All data are expressed as means ± SD. All cellular experiments were performed in at least 3 biological replicates, and all blots were normalized to β-actin. The antibody against active β-catenin recognizes the unphosphorylated, stable form of β-catenin.