According to the time point that exhibited the highest level phos phospecific of Akt and mTOR, the myotubes were treated with AS, and 1 uM wortmannin, an inhibitor of PI3K, was added for 30 min to break the PI3KAkt mTOR pathway. After incubation, the myotubes from the cell culture plate were scraped into an eppendorf selleck chemical Brefeldin A tube to analyze the protein levels of phosphorylated Akt on Ser473 Inhibitors,Modulators,Libraries and mTOR on Ser2448. This analysis was conducted using western blotting. Cells were lysed using a CelLytic Extraction Kit with 1% phosphatase in hibitor cocktail 3. Quantification was performed using a protein assay. Samples containing 50 ug of total protein were separated using sodium dodecyl sulfate polyacrylamide gel electrophor esis for 150 min at 120 V by applying 8% gradient gels on a Criterion electrophoresis cell.
Proteins were transferred to a polyvinylidene fluoride membrane at a 100 mA constant current for 10 h on ice at 4 C. The membrane was blocked in a tris buffered saline solution containing 0. 1% Tween 20 and 5% nonfat dry Inhibitors,Modulators,Libraries milk for 1 h and then incubated Inhibitors,Modulators,Libraries overnight at 4 C, using commercially available rabbit polyclonal pri mary phosphospecific antibodies. These antibodies rec ognized the phosphorylated Akt on Ser473, mTOR on Ser2448, and B actin. All antibodies were diluted to a 1 500 ratio in TBS T containing 5% nonfat dry milk. The membranes were then washed in TBS T, incubated using a secondary antibody, and diluted to a ratio of 1 16 000 in TBS T with 5% milk for 1 h, followed by washing in TBS T.
Phosphorylated proteins were visualized Inhibitors,Modulators,Libraries using enhanced chemiluminescence accord ing to the manufacturers protocols and quantified using MediaCybernetic Image Pro Plus software. The membranes described above were incubated in Restore Western Blot Stripping Buffer for 30 min and reprobed using the appropriate antibodies for detecting the total expression levels of Akt, mTOR, and B actin by using western blot analysis. All experiments were performed in triplicate. Statistical analysis All values were expressed as the Inhibitors,Modulators,Libraries mean standard devi ation. The myotube diameters of the 2 treatments were compared using a Students t test. The phosphorylation levels of Akt or mTOR at various treatment time points were analyzed using a one way analysis of variance. Group and treatment effect data were analyzed using a 2 way ANOVA combined with Scheffe posthoc analysis.
Significance was determined at the P 0. 05 level. All tests were performed using Statistical Package for Social Science soft ware Version 14. 0 for Microsoft Windows. Detection of ferulic acid in Angelica Sinensis by using high performance liquid chromatography To confirm the quality of the AS, we detected its main chemical constituents. The amount of ferulic acid in selleck inhibitor the AS was analyzed using a high performance liquid chroma tographic method.