Plin5

Muscle tissues and cell extracts were homogenized in a buffer containing 50 mM HEPES, pH 7.4, 2 mM EDTA, 150 mM NaCl, 30 mM NaPPO₄, 10 mM NaF, 1% Triton X-100, 1.5 mg/ml benzamidine HCl and 10 μl/ml of each: protease inhibitor, phosphatase I inhibitor and phosphatase II inhibitor (Sigma-Aldrich). Tissue homogenates were centrifuged for 25 min at 15,000 g and supernatants were stored at −80 °C. A total of 30 μg of solubilized proteins from muscle tissue and myotubes were run on a 4–12% SDS-PAGE (Biorad), transferred onto nitrocellulose membrane (Hybond ECL, Amersham Biosciences), and blotted with the following primary antibodies: PLIN5 (#GP31, Progen), ATGL (#2138, Cell Signaling Technology Inc.), Akt (#4691, Cell Signaling Technology Inc.), pAkt S473 (#4060, Cell Signaling Technology Inc.), pAkt T308 (#2965, Cell Signaling Technology Inc.). Subsequently, immunoreactive proteins were blotted with secondary HRP-coupled antibodies (Cell Signaling Technology Inc.) and revealed by enhanced chemiluminescence reagent (SuperSignal West Femto, Thermo Scientific), visualized using the ChemiDoc MP Imaging System and data analyzed using the ImageLab 4.2 version software (Bio-Rad Laboratories, Hercules, USA). GAPDH (#2118, Cell Signaling Technology Inc.) was used as an internal control.

Proteins were extracted and immunoblotted as described previously [22 (link)]. The following primary antibodies included FAS, ATGL, glucagon-like peptide-1 receptor (GLP-1R), phospho-ERK1/2 (p-ERK1/2), total ERK1/2, P70^S6K, Bcl-2, phospho-mTOR (p-mTOR), total mTOR which were all purchased from Cell Signaling and PLIN5 from Progen Biotechnik, ACO from Abcam, CHOP from Immunoway, CPT-1 and BiP from Santa Cruz. All the secondary antibodies and internal reference GAPDH were all purchased from Santa Cruz. Immunoreactive bands were visualized with an ECL reagent kit (Millipore). Optical densities of each band were calculated and analyzed by using Image J analysis software.