FIG 6.

Skeletal muscle IKKβ is a novel β-catenin kinase during hyperammonemia. (A) Representative immunoblotting and densitometry of active dephosphorylated and total β-catenin, cMYC, and UBF1 in C2C12 myotubes transfected with shRNAs targeting IKKβ or a scrambled shRNA (shRan) that were either untreated (UnT) or treated with 10 mM AmAc for 24 h. (B) Representative immunoblots of IKKβ and IKKα and immunoblots and densitometry of total and active β-catenin in untreated C2C12 myotubes or those treated with 10 mM AmAc for 24 h, stably expressing IKKβ or IKKα shRNA or a scrambled control (shRan). (C) Representative photomicrographs and myotube diameters of differentiated C2C12 myotubes depleted of IKKα or IKKβ or transfected with a vector with a random construct (shRan), treated with and without 10 mM AmAc. Scale bars, 40 μm. (D) Representative photomicrographs of immunofluorescence of active, dephosphorylated β-catenin expression (red) in myotubes stably expressing shRNAs targeting IKKβ or IKKα or a scrambled shRNA (shRan) that were either untreated or treated with 10 mM AmAc for 24 h. Nuclei were counterstained with DAPI (blue). Scale bars, 40 μm. (E) Luciferase activity for β-catenin transcriptional activity quantified by a Topflash assay (normalized to Renilla) in untreated C2C12 myotubes or those treated with 10 mM AmAc for 24 h, stably expressing either an shRNA targeting IKKβ shRNA or a scrambled control (shRan). All cellular experiments were performed in at least 3 biological replicates, all blots were normalized to β-actin, and data are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (shIKKβ versus shRan). The antibody against active β-catenin recognizes the unphosphorylated, stable form of β-catenin. Bars, 40 μm.