Nef1002001pk

Protein was extracted using RIPA lysis buffer (EMD Millipore Billerica, MA, USA, 10 × RIPA buffer) and quantified via the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). The protein samples were mixed with SDS loading buffer and separated on an 8–12% SDS-PAGE gel and transferred to PVDF membrane (NEF1002001PK, Perkin Elmer, Waltham, MA, USA). The membrane was then blocked with 5% BSA (Sigma) at room temperature for 1 h in Tris-buffered saline-Tween 20 (0.5%; TBS-T). After washing in TBS-T, membranes were incubated overnight at 4 °C in 2.5% BSA with the appropriate primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. All antibodies and dilutions are shown in Table S2. The immunoblot signals were developed using the Super Signal Ultra chemiluminescent reagent (Pierce, Rockford, US) and detected by a CCD camera (MultiGel-21, Topbio, Taipei, Taiwan).

Harvested cells were lysed by PRO-PREP Protein Extraction Kit (iNtRON Biotechnology, Gyeonggi-do, Korea) 5and centrifuged at 10,000 rpm at 4 °C for 30 min. The supernatant was incubated at 95 °C for 5 min in 6× sample loading buffer containing 0.35 M Tris-HCl (pH 6.8), 10% SDS, 30% glycerol, 0.12% bromophenol blue and 6% β-mercaptoethanol. About 50 μg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting to a polyvinylidene fluoride (PVDF) membrane (NEF1002001PK; Perkin Elmer, Boston, MA, USA). Non-specific binding of the blotted membrane was blocked by using 5% non-fat milk dissolved in 1× Tris-buffered saline (TBS) with 0.2% Tween 20 (0.2% TBST). The membrane was incubated with the primary antibody (in 0.2% TBST) overnight at 4 °C. Subsequently, the secondary antibody (in 0.2% TBST) was applied for 3 h at 4 °C. The expected protein bands were visualized using the ECL reaction (Amersham, Arlington Height, IL, USA), and the luminescence signal was acquired with the Fujifilm LAS-4000 system (San Leandro, CA, USA). Band intensity was analyzed using MultiGauge software (Fujifilm). The band intensity of individual proteins was normalized to that of β-actin and presented as the fold change from the control group (without the treatment of tested drugs).

Skeletal muscle (femoris and gastrocnemius) and cardiac muscle tissue samples were homogenized using a MagNA Lyser (Roche, Basel, Switzerland) in RIPA buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS with complete protease inhibitor and phosphatase inhibitor cocktails [Roche, #04693124001]) and then denatured in 2% SDS sample buffer (50 mM Tris at pH 6.8, 100 mM Dithiothreitol, 2% SDS and 10% glycerol) for 15 min at 100 °C. Total protein lysate was separated by SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and then electro-transferred to a polyvinylidene fluoride transfer membrane (PerkinElmer, Waltham, MA, USA, #NEF1002001PK). The membrane was blocked with 5% (w/v) non-fat dried milk in TBST buffer (25 mM Tris at pH 7.5, 137 mM NaCl, 2.7 mM KCl and 0.1% Tween-20 [v/v]) for 1 h at room temperature, and then incubated with a primary antibody for 14–16 h at 4 °C. The membrane was then washed three times with TBST buffer before probing with an appropriate secondary antibody for 1 h at room temperature; washing and then detection by ECL (Thermo Fischer Scientific, Waltham, MA, USA, #34580). The following antibodies were used: Cisd2, Gapdh (Millipore, Burlington, MA, USA, #MAB374), Anti-Rabbit IgG HRP Linked (Sigma-Aldrich, #NA934) and Anti-Mouse IgG HRP Linked (Sigma-Aldrich, #NA931).