Journal: bioRxiv

Article Title: The 14-3-3/spastin pathway coordinates neurite regeneration and recovery of spinal cord injury by targeting microtubule dynamics

doi: 10.1101/2023.07.25.550600

Figure Lengend Snippet: (A) Mass spectrum of peptides which was pull-downed by GST-spastin from proteins of the spinal cord tissue (near 1 cm around the T10 level), the representative peptides of 14-3-3 were shown. (B) Immunoprecipitation assay using spastin antibody was performed and Pan 14-3-3 antibody was used for western blotting. (C) Hippocampal neurons were stained with 14-3-3s (green) and spastin (red) antibodies, the growth cone (C1) and axon shaft (C2) profile were shown. (D) Localization of spastin (red line) and 14-3-3 (green line) protein in the hippocampal neurons were analyzed and the intensity of the white dotted line across the neurite compartment were measured. (E) Purified GST and GST-14-3-3s fusion proteins were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and coomassie blue staining was confirmed the successful purification of relative proteins. (F) GST fusion proteins were used to pull down the lysates of the spinal cord and spastin antibody was used for western blotting. (G) HEK 293T cells were transfected with Flag tagged 14-3-3s and GFP tagged spastin, the cell lysates were subjected to GFP-Trap assay, Flag antibody was used to detect the presence of 14-3-3s.

Article Snippet: Anti-spastin (1:1000, ABN368 from Millipore), anti-Pan 14-3-3 (1:1000, #8312 from Cell Signaling Technology), anti-GFP (1:5000, Ab290 from Abcam), anti-Flag (1:1000, F3165 from Sigma-Aldrich), anti-GAPDH (1:5000, AC002 from ABclonal), and HRP conjugated goat-derived anti-rabbit or anti-mouse were the antibodies used (1:5000, Abclonal).

Techniques: Immunoprecipitation, Western Blot, Staining, Purification, Polyacrylamide Gel Electrophoresis, Transfection, TRAP Assay

Journal: bioRxiv

Article Title: The 14-3-3/spastin pathway coordinates neurite regeneration and recovery of spinal cord injury by targeting microtubule dynamics

doi: 10.1101/2023.07.25.550600

Figure Lengend Snippet: (A) After spinal cord contusion, the spinal cord tissues of the lesion site (near 1 cm) at indicated times (1, 3, 7 14, and 30 DPI) were ground and lysed, then subjected to western blot. (B) Quantitative analysis of the 14-3-3 protein expression (n=3 per group). (C) The lesion site of spinal tissue in the sham group and the SCI group were stained with Pan 14-3-3 (red) and βIII tubulin (green). The arrows mean the elevated 14-3-3 protein in the neuronal compartment. (D) 14-3-3 agnoist FC-A and spastin inhibitor spastazoline were admisnistrated after spinal cord contusion. At 45 DPI (Days post-injury), the tissues were fixed and embedded, then cut horizontally and longitudinally. H&E and LFB (Luxol fast blue stain) were stained. The spinal cord demyelination was shown inside the area of the dotted line. Scale bar, 200 μ m. (E) The slices were then subjected to immunofluorescence staining, and GFAP (green) was stained to label astrocytes, NF (red) was stained to label neurons and 5-HT (magenta) was stained to label monoaminergic axons. The lesion site in different groups was boxed and enlarged in (E1)(E2)(E3)(E4). Scale bar, 400 μm, and 100 μm. (F) The spinal cord slices were stained with acetylated tubulin (Stable MTs which lack of dynamics) and β-tubulin (total tubulin). Scale bar, 50 μm. (G) The normalized intensity of NF was quantified (n=5 animals per group). Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. (H) The normalized ratio of acetylation tubulin to total tubulin was calculated by the intensity value of acetylated tubulin divided by total tubulin (n=5 pictures per group). Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-spastin (1:1000, ABN368 from Millipore), anti-Pan 14-3-3 (1:1000, #8312 from Cell Signaling Technology), anti-GFP (1:5000, Ab290 from Abcam), anti-Flag (1:1000, F3165 from Sigma-Aldrich), anti-GAPDH (1:5000, AC002 from ABclonal), and HRP conjugated goat-derived anti-rabbit or anti-mouse were the antibodies used (1:5000, Abclonal).

Techniques: Western Blot, Expressing, Staining, Immunofluorescence