AMPK Activation by Seaweed Extracts in C2C12 Cells

To determine the alteration of AMPK activation by seaweed extracts in C2C12 cells, Western blotting analysis was performed as described previously [19 (link)]. Briefly, the extracted proteins (30~50 μg/24 μL) from the C2C12 myotubes treated with either control or seaweed extracts were quantified. Equal amounts of proteins were loaded and separated by SDS–PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with the indicated antibody and horseradish peroxidase-coupled anti-species antibodies. The antibodies used were the following; phosphorylated AMPK (Thr172, Cell Signaling, Beverly, MA, USA, 1:1000), AMPK (Cell Signaling, Beverly, MA, USA, 1:1000), GAPDH (Cell Signaling, Beverly, MA, USA, 1:1000). Proteins were visualized using Chemidoc (Bio-Rad, Hercules, CA, USA) and quantified using Image J (National Institutes of Health, Bethesda, MD, USA).