| Agitator |
2018-01-20 22:18 |
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Materials and methods
Reagents
Antibodies against total myosin heavy chain (MHC), MHC2A,
MHC2B, and myogenin were obtained from the
Developmental Studies Hybridoma Bank, University of Iowa
(Iowa City, IA, USA). Antibodies against phospho-Akt (ser473),
Akt, p-AMPK(T172), AMPK, p-mTOR(Ser2448), mTOR, pS6
(Ser235/236), S6, pMKK3/6, MKK3, phospho-p38, and p38,
were obtained from Cell Signaling Biotechnology (Beverly, MA,
USA). GAPDH was purchased from Santa Cruz Biotechnology
(Dallas, TX, USA). PGC-1α antibody was purchased from Abcam
(Cambridge, MA, USA). Caffeine, chlorogenic acid, and caffeic
acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
HPLC-grade methanol was purchased from JT Baker Inc.
(Phillipsburg, NJ, USA). HPLC-grade water was obtained using
Sartorius water purification system (0.22 µm pore size,
Sartorius Stedim Biotech Bmbh, Germany). All other chemicals
were purchased from Sigma-Aldrich.
Sample preparation
Distilled water (80℃) was added to a commercially prepared
instant coffee powder (Dong Suh Food. Co., Seoul, Korea). For
cell culture experiments, the solution was filtered through a
membrane filter under a vacuum.
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