Rabbit phospho ampkα thr172

Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Primary antibodies were: rabbit phospho-AMPKα (Thr172) (1:1000; Cell Signaling Technology, USA), mouse anti-AMPK (1:1000; Proteintech, USA), rabbit anti-Bax (1:1000; Cell Signaling Technology, USA), rabbit anti-Bcl-2 (1:500; Cell Signal Technology, USA), rabbit anti-Sirtin1(1:1000, Millipore, USA), rabbit anti-COX2 (1:1000; Abcam, USA), rabbit anti-GFAP (1:1000; Dako, Japan), mouse anti-β-actin (1:1000; Santa Cruz Biotechnology, USA), mouse anti-NF-κB p65 (1:1000; Santa Cruz Biotechnology, USA), and rabbit anti-phospho-NFκB p65 (1:1000; Cell Signaling Technology, USA). Protein bands were detected with an Odyssey infrared imaging system (Li-Cor, USA).

OPCs and OLs cells were lysed in a 20 mM Tris and 1% sodium dodecyl sulfate (SDS) pH 7.4 solution pre-heated to 95°C. Using a cell scraper, lysate was collected into microfuge tubes and placed on a 95°C heating block for 10 min. Protein concentration was determined following the manufacturer’s instructions of the Bio-Rad DC Assay (Biorad). Protein lysates were electrophoresed on 10 or 12% SDS polyacrylamide gels (Biorad). After SDS-PAGE, proteins were transferred to a 0.45 μm nitrocellulose membrane (GE Healthcare) using western blotting, and blocked with a 4% bovine serum albumin (Sigma-Aldrich) in 1x tris buffered saline +0.1% tween (Sigma-Aldrich) solution (TBST). After blocking, the membrane was incubated overnight with the following primary antibodies: 1:750 rabbit AMPKα (Cell Signaling, 5,831), 1:750 rabbit phospho-AMPKα (Thr172) (Cell Signaling, 2,531), 1:750 rabbit ACC (Cell Signaling, 3,676), 1:750 rabbit phospho-ACC (Ser79) (Cell Signaling, 11,818), and 1:750 rabbit Histone H3 (Santa Cruz Biotenchnology, FL-136). The next day the primary antibodies were washed 3 times with 1x TBST. Next, membranes were incubated in 1x TBST for 1 h with 1:2000 anti-rabbit horseradish peroxidase linked secondary antibodies (Cell Signaling, 7,074). Enhanced chemiluminescence substrate kit (Millipore) was used to develop protein blots on radiography films (Thermo Scientific).