|Remove medium and rinse cells in 1X PBS.
Add 1X lysis reagent.
Mix 20µl of cell lysate with 100µl of Luciferase Assay Reagent and measure the light produced.
The FLAG-SIRT1 and FLAG-SIRT1 H355A genes were kindly provided by Professor Soo-Jong Um at Sejong University, and the myogenin- and creatine kinase promoter-luciferase reporter genes were provided by Professor Jeong-Ho Hong at Korea University. The mitsugumin 53 (MG53) promoter-luciferase reporter gene was previously generated (40). Gene transfection was performed using electroporation according to the protocol of electroporator MP-100 (Invitrogen) for C2C12 myoblasts. Luciferase activity was measured by using the luciferase assay system (Promega) using a Luminoskan Ascent (Thermo Labsystems). We normalized the luciferase activity to the activity of coexpressed β-galactosidase. To calculate the relative luciferase activity, we divided each normalized luciferase activity by that of the empty vector-transfected cells. Full paper
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