Mitochondrial isolation, protein extraction and western blot analysis

The ipsilateral side of the injured spinal cord segments (5 mm) were dissected out after 7 DPI and homogenized in ice-cold lysis buffer containing 1 × protease inhibitor cocktail and phosphatase inhibitors. Mitochondria were isolated using the Mitochondria Isolation Kit (Biochain. Catalog# {"type":"entrez-nucleotide","attrs":{"text":"KC010100","term_id":"529515746","term_text":"KC010100"}}KC010100, Newark, CA). The protein concentration from the tissue homogenates and isolated mitochondria was determined using a BCA protein assay kit (Pierce). The extracted proteins were analyzed in polyacrylamide-SDS gels and electro-blotted using PVDF membranes. Some of the membranes were cut prior incubation with the antibodies. After blocking, the membranes were probed overnight at 4ºC with the rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), anti-Total OXPHOS Antibody Cocktail, anti-p44/42 MAPK (ERK1/2), anti-STAT3, anti-STAT3-Ser727, anti-VDAC1, anti-phospho-NRF2, anti-TFAM, anti-mTOR, anti-phospho-mTOR (Ser2448), anti-phospho-p4E-BP1, anti-Actin, anti-phospho-p70-SK6. Secondary fluorescent antibodies IRDye (800, 680CW IgG (H + L) (1:15,000, LI-COR) were applied to the membrane and incubated for 1 h at room temperature. Blots were visualized using Odyssey Fc Imaging System (LI-COR, Nebraska, USA). Densitometric analysis was performed using Image Studio Lite Ver 5.2. For more detailed information about the antibodies used, see Table of Antibodies (Table ​(Table2)2) at the end of the “Methods” section.

List of antibodies, vendors and used dilutions.