Antimouse total oxphos

The cells were washed twice with ice-cold PBS and lysed in Cell Lysis Buffer (1x). Protein levels were determined using the Bradford method, and then the samples were mixed with Laemmli buffer and denatured at 95°C for 5 min. Equal amounts of proteins were separated on SDS/PAGE gels. All proteins were transferred to nitrocellulose membranes at 100 V. Membranes were washed for 5 min in TBS-Tween buffer (0.1% TBST) (100 mM Tris-buffered saline, 140 mM NaCl, and 0.1% Tween 20; pH 7.6) and the nonspecific bindings were blocked for 1 h at RT with 5% BSA in 0.1% TBST or with 5% nonfat milk solution in 0.1% TBST. Immunodetection was performed overnight at 4 °C using rabbit antiparkin (1:500; Cell Signaling), rabbit anti-AMPK (1:1000, Cell Signalling), rabbit anti-p-AMPK (1:1000, Cell Signalling), rabbit anti-Ulk-1 (1:200, Cell Signalling), rabbit anti-p-Ulk-1 (1:200, Cell Signalling), and antimouse total OXPHOS (1:500, Abcam) antibodies. Then, the membranes were washed three times (5 min) in TBST and incubated for 60 min at RT with antirabbit or antimouse secondary antibody (1∶4000) in a 5% nonfat milk/TBST. Antibodies were detected using chemiluminescent Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) under standard conditions. Immunolabeling of GAPDH (rabbit anti-GAPDH; 1:40,000; Sigma-Aldrich) for cell lysates was performed as a loading control.

Cultured cells were washed with cold PBS before homogenization in lysate buffer. Whole cell lysate (20 μg protein/sample) was used in Western blot analysis. Cell lysates were separated by electrophoresis on 10–12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5 % non-fat milk/PBS with Tween 20 for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies, including mouse anti-human CD133 (Miltenyi Biotec), rabbit anti-human SOX2 (Cell Signaling Technology Inc., Danvers, MA, USA), rabbit anti-human Olig2 (Abcam, Cambridge, MA, USA), rabbit anti-human Catalase (EMD Chemicals, Gibbstown, NJ, USA), sheep anti-human SOD1 (EMD Chemicals), rabbit anti-human SOD2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and anti-mouse total OXPHOS (Abcam). The Western blot signals were detected with horseradish peroxidase-conjugated secondary antibodies. The membranes were developed by using a Pierce Supersignal West Pico Chemiluminescent Substrate (Fisher Scientific Inc., Pittsburgh, PA, USA).