Protease inhibitor cocktail

The samples were disrupted using a homogenizer, after which an ice-cold RIPA protein extraction solution with a protease inhibitor cocktail (iNtRON biotechnology, Inc., Seoul, Korea) was added, and the samples were homogenized with stainless steel beads (Qiagen, Cam, USA). Protein concentrations were assessed using a BCA-kit (Thermo Scientific, Rockford, IL, USA). An equal amount of protein (50 μg) from each sample was loaded onto 10% to 12% SDS gel, and transferred to a PVDF membrane (Merk Millipore, MA, USA). The membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween-20 and incubated with anti-bodies (1:1000) overnight at 4°C (Suppl. Table 1). After washing three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) at RT for 2 h and visualized with a chemiluminescence substrate. Quantitative analysis of the western blot images was performed using ImageJ Software.

The frozen samples were disrupted using the TissueLyser II (Qiagen), after which an ice-cold PRP-PREP protein extraction solution with a protease inhibitor cocktail (iNtRON Biotechnology, Inc., Seoul, Korea) was added, and the samples were homogenized by stainless steel beads (Qiagen, Cam USA). Protein concentration was assessed using the BCA-kit (Thermo Scientific, Rockford, IL, USA). An equal amount of protein (80 μg) from each sample was loaded onto 10 to 12% SDS gel, and transferred to a PVDF membrane (Merk Millipore, MA, USA). The membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween-20, and incubated with primary antibodies (1:1000 and 1:500) overnight at 4 °C. After washing three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) at RT for 2 h and visualized with a chemiluminescence substrate.

Ice-cold PRP-PREP protein extraction solution with protease inhibitor cocktail (iNtRON Biotechnology, Inc, Seoul, Korea) was added after FIR treatment of the samples, followed by homogenization using stainless steel beads (Qiagen). An equal amount of protein (50 μg) for each sample was loaded onto a 10 - 12% SDS gel, subjected to electrophoresis, and transferred to the PVDF membranes (Merk Millipore, MA, USA). The membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween-20, and incubated with primary antibodies (1:1000 and 1:500, respectively) overnight at 4°C. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) at RT for 2 h, and then visualized with a chemiluminescence substrate.

The frozen samples were disrupted using the TissueLyser II (Qiagen), after which an ice-cold PRP-PREP protein extraction solution with a protease inhibitor cocktail (iNtRON Biotechnology, Inc., Seoul, Korea) was added, and the samples were homogenized with stainless steel beads (Qiagen, CA, USA). Protein concentration was assessed using a BCA kit (Thermo Scientific, Rockford, IL, USA). An equal amount of protein (80 μg) from each sample was loaded onto 10% to 12% SDS gel and transferred to a PVDF membrane (Merck Millipore, MA, USA). The membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in PBS containing 0.1% Tween-20 and incubated with primary antibodies (1 : 1000 and 1 : 500, respectively) overnight at 4°C. After washing three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 5000) at RT for 2 h and visualized with a chemiluminescence substrate.

Proteins were extracted from SN4741 cells using RIPA lysis buffer (100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM EDTA, 0.2% SDS, phosphatase, and a protease inhibitor cocktail) (iNtRON Biotechnology, Gyeonggi, South Korea). After centrifugation at 15000×g for 20 min at 4°C, supernatants were collected. Protein levels were measured using the Bradford (Bio-Rad, CA, USA) method. Isolated proteins (20 μg) were resolved using 10-12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF, Millipore, ISEQ00010) membranes, which were blocked with 5% BSA in TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20). The membranes were incubated overnight at 4°C with primary antibodies against NDUFA9 (Invitrogen, 459100), NDUFA8 (Invitrogen, 459210), SDHA (Invitrogen, 459200), UQCRC2 (Invitrogen, A11143), COX4 (Invitrogen, A21348), ATP5A1 (Invitrogen, A21350), Mn SOD (MA1-106), Cu-Zn SOD (Novus Biologicals, NBP2-24915), HSP60 (Santa Cruz, sc-1052), and actin (Santa Cruz, sc-8432) and then with a horseradish peroxidase-coupled secondary antibody for 1 h at room temperature (RT). Finally, the antibody-labeled proteins were detected using an ECL system (iNtRON Biotechnology, Gyeonggi, South Korea). All antibodies were validated by the manufacturer. Band quantification was performed with the ImageJ software (http://imagej.nih.gov/ij; v.1.47b).