Eif5a1

Section preparation and staining were carried out as described previously17 (link). Briefly, after the final BBB evaluation, animals were perfused with 4% paraformaldehyde solution. Spinal cord tissue (0.5 cm caudal to the injury) was harvested and placed in 4% paraformaldehyde then successively in 0.1 M phosphate buffered saline solutions that contained 10%, 20% and 30% sucrose. Spinal cord tissue was sectioned at 20 μm thickness in a freezing microtome (Leica CM1900, Germany). Every 50 sections, 1 slide was selected for immunofluorescence staining. After blocking for 1 hr with 10% normal goat serum, sections were incubated in primary antibody (eIF5A1, 1:200, Abcam; RhoGDIα, 1:200, Abcam; SMI-312(NF), 1:500, Covance; NeuN, 1:400, Abcam). Goat anti-rabbit (1:400, Invitrogen) and goat anti-mouse (1:400, Invitrogen) were used as secondary antibody. Lastly, sections were imaged with Leica AF6000 fluorescence microscope. The neurofilament positive cells (NF+) and NeuN positive cells (NeuN+) were measured by Leica LAS AF software. We used this software to compute the positive cells in spinal gray matter per mm². The computing method of Fig. 3E,F and Fig. 6 D,E is following. Positive cells (NF+ or NeuN+) in total (%) = (positive cells number/total cells number (DAPI + nucleus)/area (mm²))*100.

Protein extraction and immunoblotting were performed as described by Shang et al.23 (link). Antibodies directed against the following proteins were used: eIF5A1 (1:5000, Abcam, USA), phosphorylated map2k6 (1:1000, Abcam, USA), and β-tubulin loading ctrl (β-TUB, 1:2000, GeneTex, USA). HRP-conjugated secondary antibodies (1:5000, GeneTex, USA) were also used. Blotting was analyzed using ImageJ software. And each sample was normalized to β-TUB.