Journal: Neuroscience letters

Article Title: Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

doi: 10.1016/j.neulet.2021.136124

Figure Lengend Snippet: Fig. 3. The expression of miR-338-5p was lower in the rats with SCI than the control rats, and the miR- 338-5p-overexpressing exosomes provided neuro protective effects after SCI. (A) Expression of miR- 338-5p was assessed by qRT-PCR at 1, 4 and 7 days after acute spinal cord injury in rats. (B, C) Western blot analysis of NF-M, GAP43, MAG and GFAP levels following various treatments at day 4 post-SCI. Data are presented as the mean ± SD. n = 4 per group (A), n = 3 per group (C), #p < 0.05, ##p < 0.01, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, they were incubated with primary antibodies overnight at 4◦C as follows: TSG101 (1:1000, Santa Cruz Biotechnology), HSP70 (1:1000, Santa Cruz Biotechnology), CD63 (1:1000, Santa Cruz Biotechnology), CD9 (1:1000, Santa Cruz Biotechnology), Cnr1 (1:1000, Santa Cruz Biotechnology), Rap1 (1:1000, Santa Cruz Biotechnology), neurofilament-M (NF-M) (1:1000, Proteintech Group), growth associated protein-43 (GAP43) (1:1000, Santa Cruz Biotechnology), myelinassociated glycoprotein (MAG) (1:1000, Proteintech Group), glial fibrillary acidic protein (GFAP) (1:1000, Proteintech Group), Bax (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology), pro caspase-3 (1:1000, Cell Signaling Technology), cleaved-caspase-3 (1:1000, Cell Signaling Technology), total-PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), total-AKT (1:1000, Cell Signaling Technology), and p-AKT (1:1000, Cell Signaling Technology).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Neuroscience letters

Article Title: Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

doi: 10.1016/j.neulet.2021.136124

Figure Lengend Snippet: Fig. 4. Immunofluorescence double staining of NF-M, GAP43, MAG and GFAP levels following various treatments at day 4 post-SCI in rats. (A) Increased levels of the NF-M and GAP43 proteins in the region of anterior horn and adjacent lateral funiculus were observed in the miR-338-5p exo group compared to the other SCI groups at day 4 post-SCI. (B) Decreased protein levels of MAG and GFAP in the region of anterior horn and adjacent lateral funiculus were observed in the miR-338-5p exo group compared to the other SCI groups at day 4 post-SCI. Scale bar, 200 µm. (C) Semi-quantification of the mean fluorescence intensity of NF-M, GAP43, MAG and GFAP were measured. Data are presented as the mean ± SD. n = 3 per group, #p < 0.05, ##p < 0.01, ###p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, they were incubated with primary antibodies overnight at 4◦C as follows: TSG101 (1:1000, Santa Cruz Biotechnology), HSP70 (1:1000, Santa Cruz Biotechnology), CD63 (1:1000, Santa Cruz Biotechnology), CD9 (1:1000, Santa Cruz Biotechnology), Cnr1 (1:1000, Santa Cruz Biotechnology), Rap1 (1:1000, Santa Cruz Biotechnology), neurofilament-M (NF-M) (1:1000, Proteintech Group), growth associated protein-43 (GAP43) (1:1000, Santa Cruz Biotechnology), myelinassociated glycoprotein (MAG) (1:1000, Proteintech Group), glial fibrillary acidic protein (GFAP) (1:1000, Proteintech Group), Bax (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology), pro caspase-3 (1:1000, Cell Signaling Technology), cleaved-caspase-3 (1:1000, Cell Signaling Technology), total-PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), total-AKT (1:1000, Cell Signaling Technology), and p-AKT (1:1000, Cell Signaling Technology).

Techniques: Immunofluorescence, Double Staining, Fluorescence

Journal: Neuroscience letters

Article Title: Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

doi: 10.1016/j.neulet.2021.136124

Figure Lengend Snippet: Fig. 6. Overexpression of miR-338-5p alleviated H2O2-induced cell injury in PC12 cells. (A) PC12 cells were transfected with NC mimics/inhibitors, miR-338-5p mimics and inhibitors. qRT-PCR analysis of the miR-338-5p level. (B, C) Measurement of intracellular ROS and SOD. (D) CCK-8 assay for cell viability. (E, F) Western blot analysis of NF-M, GAP43, MAG and GFAP levels following various treatments in PC12 cells. n = 3 per group, #p < 0.05, ##p < 0.01, ###p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, they were incubated with primary antibodies overnight at 4◦C as follows: TSG101 (1:1000, Santa Cruz Biotechnology), HSP70 (1:1000, Santa Cruz Biotechnology), CD63 (1:1000, Santa Cruz Biotechnology), CD9 (1:1000, Santa Cruz Biotechnology), Cnr1 (1:1000, Santa Cruz Biotechnology), Rap1 (1:1000, Santa Cruz Biotechnology), neurofilament-M (NF-M) (1:1000, Proteintech Group), growth associated protein-43 (GAP43) (1:1000, Santa Cruz Biotechnology), myelinassociated glycoprotein (MAG) (1:1000, Proteintech Group), glial fibrillary acidic protein (GFAP) (1:1000, Proteintech Group), Bax (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology), pro caspase-3 (1:1000, Cell Signaling Technology), cleaved-caspase-3 (1:1000, Cell Signaling Technology), total-PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), total-AKT (1:1000, Cell Signaling Technology), and p-AKT (1:1000, Cell Signaling Technology).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, CCK-8 Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

doi: 10.1083/jcb.200507061

Figure Lengend Snippet: Ral regulates GAP-43 phosphorylation. (A) COS cells were transfected with the indicated myc-tagged Ral constructs and cGAP-43. Levels of phospho-cGAP-43 in cell extracts were determined by immunoblotting with an anti–phospho-GAP-43 antibody. A representative blot is shown. Inactive Ral causes a significant decrease of cGAP-43 phosphorylation (top band, arrow). (B) Quantitative analysis of phosphorylated cGAP-43 in COS cells transfected with cGAP-43 and the indicated Ral constructs. Shown here are data from four independent experiments (means ± SEM; *, P < 0.01). (C) Cortical neurons were nucleofected with the indicated constructs and lysed after overnight expression. Levels of total and phosphoendogenous GAP-43 were then assessed by immunoblotting. The fourth, fifth, and sixth lanes were derived from the same blot. (D) Quantitative analysis of endogenous GAP-43 phosphorylation in cortical neurons after nucleofection with the indicated Ral mutant isoforms. Data were derived from four independent experiments (means ± SEM; **, P < 0.005).

Article Snippet: Antibodies used were mouse anti-RalA, rabbit anti-RalB (BD Biosciences), mouse anti–GAP-43 (Autogen Bioclear), rabbit anti–phospho(Ser41)-GAP-43 (Zymed Laboratories), rat anti–α-tubulin (Harlan), rat anti-HA (Roche), mouse anti-myc (clone 9E10), rabbit anti-myc (Research Diagnostics, Inc.), mouse anti–βIII tubulin (Sigma-Aldrich), and rabbit anti-p38 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Construct, Western Blot, Expressing, Derivative Assay, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

doi: 10.1083/jcb.200507061

Figure Lengend Snippet: GAP-43 acts downstream of Ral. (A) Constitutively active Ral increases branching in neurons plated on laminin (top middle and right) compared with EGFP-F–expressing cells (top left). This effect is reduced by coexpression of cGAP-43(S42A) (middle and bottom). Shown here are cells stained with anti-myc antibody to detect mutant Ral and anti–cGAP-43. Bar, 100 μm. (B) Quantitative analysis of branching in cells injected with single Ral isoforms or with Ral and cGAP-43(S42A) (means ± SEM: GFP, 3.30 ± 0.18; RalA72L, 8.43 ± 0.97; RalA72L + cGAP-43(S42A), 3.55 ± 0.44; RalB23V, 8.88 ± 0.85; RalB23V + cGAP-43(S42A), 4.29 ± 0.41; *, P < 0.02; **, P < 0.0001).

Article Snippet: Antibodies used were mouse anti-RalA, rabbit anti-RalB (BD Biosciences), mouse anti–GAP-43 (Autogen Bioclear), rabbit anti–phospho(Ser41)-GAP-43 (Zymed Laboratories), rat anti–α-tubulin (Harlan), rat anti-HA (Roche), mouse anti-myc (clone 9E10), rabbit anti-myc (Research Diagnostics, Inc.), mouse anti–βIII tubulin (Sigma-Aldrich), and rabbit anti-p38 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Staining, Mutagenesis, Injection

Journal: The Journal of Cell Biology

Article Title: Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

doi: 10.1083/jcb.200507061

Figure Lengend Snippet: GAP-43 acts downstream of Ral. (A) A phosphomimetic GAP-43 protein, cGAP-43(S42D), restores branching in neurons plated on laminin and expressing dominant-negative Ral. Shown here are neurons expressing EGFP-F (top left), RalA28N (top middle), or RalB28N (bottom left) and RalA28N or RalB28N and cGAP-43(S42D) (right). Cells are stained with an anti-myc antibody to detect mutant Ral and an anti–cGAP-43 antibody. Bar, 50 μm. (B) Quantitative analysis of branching in cells plated on laminin and injected with single Ral isoforms or with Ral and cGAP-43(S42D) (means ± SEM: GFP, 3.47 ± 0.37; RalA28N, 1.74 ± 0.52; RalA28N + cGAP-43(S42D), 2.42 ± 0.31; RalB28N, 1.56 ± 0.41; RalB28N + cGAP-43(S42D), 3.72 ± 0.35; *, P < 0.04; **, P < 0.001). (C) SCG neurons were plated on polyornithine, microinjected, and left to express the indicated proteins for 5 h before laminin addition. Expression of dominant-negative Ral decreases branching compared with EGFP-F–expressing cells (top, compare middle and right with left). Branching is restored in cells coexpressing cGAP-43(S42D) (middle and bottom rows). Bar, 100 μm. (D) Quantitative analysis of branching in neurons initially plated on polyornithine and injected with single Ral isoforms or with Ral and cGAP-43(S42A) before laminin addition (means ± SEM: GFP, 2.64 ± 0.35; RalA28N, 1.05 ± 0.32; RalA28N + cGAP-43(S42D), 1.82 ± 0.40; RalB28N, 1.74 ± 0.30; RalB28N + cGAP-43(S42D), 2.73 ± 0.45; **, P < 0.01).

Article Snippet: Antibodies used were mouse anti-RalA, rabbit anti-RalB (BD Biosciences), mouse anti–GAP-43 (Autogen Bioclear), rabbit anti–phospho(Ser41)-GAP-43 (Zymed Laboratories), rat anti–α-tubulin (Harlan), rat anti-HA (Roche), mouse anti-myc (clone 9E10), rabbit anti-myc (Research Diagnostics, Inc.), mouse anti–βIII tubulin (Sigma-Aldrich), and rabbit anti-p38 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Dominant Negative Mutation, Staining, Mutagenesis, Injection