2.8. Western blot analysis

On day 3 after SCI, long spinal cord segments (1.0 cm, in length) at the injury epicenter of rats (n = 5) in each group were dissected and immediately stored at -80°C for further Western blot analysis as previously published [42]. After protein extraction from the flap homogenate, the protein concentration was determined via a BCA test kit. 55 μg protein was separated on a 12% (w/v) gel and transferred onto PVDF membranes. After blocking with 10% nonfat milk, the membranes were incubated with the following primary antibodies at 4°C overnight: mTOR (1 : 1000), p-mTOR (1 : 1000), p70S6K (1 : 1000), p-p70S6K (1 : 1000), LC3B (1 : 500), Beclin1 (1 : 1000), p62 (1 : 500), CTSD (1 : 500), ubiquitin (1:1000), PARP (1 : 1000), Bax (1 : 1000), cleaved CASP3 (1 : 400), cytochrome c (1 : 1000), and β-actin (1 : 1000). After that, the membranes were reincubated with the HRP-conjugated IgG secondary antibody (1 : 5000) at room temperature for 1.5 h. Then, bands on the membranes were visualized via the ECL-Plus Immune-Detection Kit. According to an established protocol [43], the intensity of the blot was quantified by densitometry using the C-DiGit® Blot Scanner and software (LI-COR Biosciences, USA) and was normalized to the β-actin band.