Notch1 and Akt Signaling Pathway Analysis

Proteins were isolated from muscle specimens by homogenization for 30 min in ice-cold lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.25% SDS, pH 7.4)-supplemented protease inhibitor cocktail (1 tablet in 10 mL), 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride as described in [17 (link)]. Equal amounts (40 μg) of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride membrane. The blots were incubated with primary antibodies: total Notch1, cleaved Notch1 (c-Notch1), total Akt, phosphor-Akt (p-Akt), and β-actin purchased from Cell Signaling Technology (Boston, MA). Vascular endothelial growth factor (VEGF) was purchased from Santa Cruz Biotechnology.