Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Levetiracetam Protects Against Cognitive Impairment of Subthreshold Convulsant Discharge Model Rats by Activating Protein Kinase C (PKC)-Growth-Associated Protein 43 (GAP-43)-Calmodulin-Dependent Protein Kinase (CaMK) Signal Transduction Pathway

doi: 10.12659/MSM.913542

Figure Lengend Snippet: The primer sequences for the real-time PCR.

Article Snippet: The PVDF membranes were incubated with rabbit anti-rat NCAM polyclonal antibody (Catalogue No: GTX133217, 1: 2000, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), mouse anti-rat GAP43 monoclonal antibody (Catalogue No. GTX34384, 1: 3000, GeneTex, Inc.), mouse anti-rat PSD-95 monoclonal antibody (Catalogue No. MABN68, 1: 3000, Millipore, Boston, MA, USA), mouse anti-rat CaMK II monoclonal antibody (Catalogue No. sc-5306, 1: 3000, Santa Cruz Biotech), and mouse anti-β actin monoclonal antibody (Catalogue No. GTX11003, 1: 3000, GeneTex Inc.) for 2 h at room temperature.

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Levetiracetam Protects Against Cognitive Impairment of Subthreshold Convulsant Discharge Model Rats by Activating Protein Kinase C (PKC)-Growth-Associated Protein 43 (GAP-43)-Calmodulin-Dependent Protein Kinase (CaMK) Signal Transduction Pathway

doi: 10.12659/MSM.913542

Figure Lengend Snippet: The signal pathway of PKC-GAP43-CaMK.

Article Snippet: The PVDF membranes were incubated with rabbit anti-rat NCAM polyclonal antibody (Catalogue No: GTX133217, 1: 2000, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), mouse anti-rat GAP43 monoclonal antibody (Catalogue No. GTX34384, 1: 3000, GeneTex, Inc.), mouse anti-rat PSD-95 monoclonal antibody (Catalogue No. MABN68, 1: 3000, Millipore, Boston, MA, USA), mouse anti-rat CaMK II monoclonal antibody (Catalogue No. sc-5306, 1: 3000, Santa Cruz Biotech), and mouse anti-β actin monoclonal antibody (Catalogue No. GTX11003, 1: 3000, GeneTex Inc.) for 2 h at room temperature.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Mycobacterium smegmatis Induces Neurite Outgrowth and Differentiation in an Autophagy-Independent Manner in PC12 and C17.2 Cells

doi: 10.3389/fcimb.2018.00201

Figure Lengend Snippet: Western blotting analysis of differentiation-related markers of PC12 cells and C17.2 cells treated with culture supernatant of M. smegmatis . (A) PC12 cells and C17.2 cells were treated with/without culture supernatant of M. smegmatis , differentiation related markers GAP43 (Growth Associated Protein 43), SYN (Synaptophysin) and TUBB3 (Tubulin beta-3 chain) were determined by western blot analysis; (B) Neuron related markers Nestin and MAP-2 were determined by western blot analysis in C17.2 cells.

Article Snippet: After being blocked with 5% non-fat dry milk in PBS containing 0.05% Tween-20, the blotted membranes were incubated with anti-GAP43 antibody, anti-TUBB3 antibody, anti-synaptophysin antibody (1:1,000 respectively, BS3655, BS1345, AP0013, Bioworld, China), anti-Nestin antibody, anti-MAP-2 antibody, anti-P62 antibody (1:1,000 respectively, Abcam, USA), anti-LC3 antibody (1:1,000 respectively, AF5384, AF5402, Affinity, USA) and anti-Phospho-Akt antibody, anti-Akt antibody, anti-Phospho-mTOR antibody, anti-mTOR antibody (1:1,000 respectively, 9,271, 9,272, 2,971, 2,972, Cell Signaling Technology, USA) and secondary antibody (1:5,000, Boster, China). β-tubulin protein levels were also determined by using the specific antibody (1:1,000, BS1842, Bioworld, China) as a loading control.

Techniques: Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Mycobacterium smegmatis Induces Neurite Outgrowth and Differentiation in an Autophagy-Independent Manner in PC12 and C17.2 Cells

doi: 10.3389/fcimb.2018.00201

Figure Lengend Snippet: Immunofluorescence detection of differentiation markers in PC12 cells and C17.2 cells treated with culture supernatant of M. smegmatis . PC12 cells and C17.2 cells were treated with 10% M. smegmatis culture supernatant for 48 h Cells were immunofluorescence stained with GAP43 and SYN. Nuclear counterstaining was done with DAPI. Fluorescence was visualized with a laser-scanning confocal microscope (40 ×).

Article Snippet: After being blocked with 5% non-fat dry milk in PBS containing 0.05% Tween-20, the blotted membranes were incubated with anti-GAP43 antibody, anti-TUBB3 antibody, anti-synaptophysin antibody (1:1,000 respectively, BS3655, BS1345, AP0013, Bioworld, China), anti-Nestin antibody, anti-MAP-2 antibody, anti-P62 antibody (1:1,000 respectively, Abcam, USA), anti-LC3 antibody (1:1,000 respectively, AF5384, AF5402, Affinity, USA) and anti-Phospho-Akt antibody, anti-Akt antibody, anti-Phospho-mTOR antibody, anti-mTOR antibody (1:1,000 respectively, 9,271, 9,272, 2,971, 2,972, Cell Signaling Technology, USA) and secondary antibody (1:5,000, Boster, China). β-tubulin protein levels were also determined by using the specific antibody (1:1,000, BS1842, Bioworld, China) as a loading control.

Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: ACS Omega

Article Title: Constraint-Induced Movement Therapy Modulates Neuron Recruitment and Neurotransmission Homeostasis of the Contralesional Cortex to Enhance Function Recovery after Ischemic Stroke

doi: 10.1021/acsomega.4c02537

Figure Lengend Snippet: CIMT promoted expression of synaptic associated proteins after ischemic stroke. (A–L) A representative band and quantitative analysis show the expression of (A, B) PSD95, (C, D) GAP43, (E, F) SYN, and (G, H) CREB by Western blot. (I, J) The mRNA expression levels of (I) VEGF and (J) BDNF were quantified by PCR. Data are expressed as the mean ± SD ( n = 4–6). (K) The expression of cfos in the contralesional cortex is assessed by IF. (L) Quantitative analysis showing the expression of cfos in the contralesional contralesional cortex. Data are analyzed by one-way ANOVA followed by Bonferroni post hoc. Asterisks indicate significances: * p < 0.05, ** p < 0.01, and *** p < 0.001. CIMT: constraint-induced movement therapy, PSD-95: postsynaptic density 95, CREB: cAMP-response element binding protein, SYN: synaptophysin, VEGF: vascular endothelial growth factor, BDNF: brain-derived nerve growth factor.

Article Snippet: We then prepared the primary antibodies: anti-PSD 95 (1:1000, cell signal technology), anti-GAP43 (1:2000, abmart), anti-SYN (1:500, proteintech), vesicular GABA amino acid transporter(VGAT) (1:1000, proteintech), Glutamic acid decarboxylase 65 (GAD65, 1:3000, proteintech), Tubulin (1:5000; abmart), and HTR2C (1:1000; abmart).

Techniques: Expressing, Western Blot, Binding Assay, Derivative Assay

Journal: Molecular Medicine Reports

Article Title: Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats

doi: 10.3892/mmr.2015.4132

Figure Lengend Snippet: Overexpression of GAP-43 promotes the differentiation of BMSCs into neuron-like cells. (A) Western blot analysis demonstrated that the protein expression of the GAP-43 was significantly increased in the LV5-GAP-43 group, compared with the NC and Blank groups. (B) BMSCs were observed using microscopy and it was revealed that BMSCs exhibited a neuronal phenotype following transduction with LV5-GAP-43. (C) BMSCs expressed positive specific neural markers for NSE, NF, nestin and βIII-tubulin in the LV5-GAP-43 group, determined using semi-quantitative polymerase chain reaction detection. Scale bar=50 µ m. GAP-43, growth-associated protein-43; BMSCs, bone marrow mesenchymal stem cells; NSE, neuron-specific enolase; NF, neurofilament; NC, transduced with negative control scramble lentiviral vector; LV5-GAP-43, transduced with LV5-GAP-43; M, marker.

Article Snippet: The samples were then incubated with rabbit anti-rat nestin, NeuN and GAP-43 polyclonal antibodies (Wuhan Boster Biological Technology, Ltd.) at a dilution of 1:100 in PBS at 4°C overnight, washed twice with PBS and incubated with FITC- or Cy3-labeled rabbit anti-goat IgG (H+L; Wuhan Boster Biological Technology, Ltd.) at a dilution of 1:200 in PBS at room temperature for 1 h. Finally, images were captured using an inverted fluorescence microscope (Olympus Corp.).

Techniques: Over Expression, Western Blot, Expressing, Microscopy, Transduction, Real-time Polymerase Chain Reaction, Negative Control, Plasmid Preparation, Marker

Journal: Molecular Medicine Reports

Article Title: Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats

doi: 10.3892/mmr.2015.4132

Figure Lengend Snippet: Ability of BMSCs to differentiate into neuron-like cells is significantly weakened when the expression of GAP-43 in BMSCs is inhibited. (A) Western blot analysis demonstrated that LV3-shRNA-GAP-43-619 was the most effective siRNA against GAP-43. 1, transduced with LV5-GAP-43; 2, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-844; 3, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-1215; 4, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-678; 5, transduced with LV5-GAP-43 and LV3-shRNA-GAP-43-619. (B) Western blot analysis revealed that the protein expression of the GAP-43 was significantly decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC and Blank groups. (C) BMSCs exhibited a neuronal phenotype in the NC and Blank groups; however, the majority of BMSCs exhibited no marked morphological changes. (D) Western blot analysis demonstrated that the expression levels of NSE, NF, nestin and βIII-tubulin were decreased in the LV3-shRNA-GAP-43-619 group compared with NC and Blank groups. (E) Cell immunofluorescence analysis revealed that the expression of GAP-43, nestin and NeuN were decreased in the LV3-shRNA-GAP-43-619 group, compared with the NC group under induction conditions. Scale bar=50 µ m. * P<0.05, vs. Blank group; # P<0.05, vs. NC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; LV3-shGAP-43, transduced with LV3-shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; siRNA, small interfering RNA; NSE, neuron-specific enolase; NF, neurofilament; NeuN, neuron-specific nuclear-binding protein.

Article Snippet: The samples were then incubated with rabbit anti-rat nestin, NeuN and GAP-43 polyclonal antibodies (Wuhan Boster Biological Technology, Ltd.) at a dilution of 1:100 in PBS at 4°C overnight, washed twice with PBS and incubated with FITC- or Cy3-labeled rabbit anti-goat IgG (H+L; Wuhan Boster Biological Technology, Ltd.) at a dilution of 1:200 in PBS at room temperature for 1 h. Finally, images were captured using an inverted fluorescence microscope (Olympus Corp.).

Techniques: Expressing, Western Blot, shRNA, Transduction, Immunofluorescence, Negative Control, Plasmid Preparation, Small Interfering RNA, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats

doi: 10.3892/mmr.2015.4132

Figure Lengend Snippet: Intravitreally injected BMSCs overexpressing GAP-43 promote the nerve repair process in a rat model of TON. (A) Fluorogold staining revealed that the number of labeled RGCs was decreased in the TON model rats, compared with normal rats at day 7. (B) Number of labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction as the duration extended in TON model rats, compared with the normal rats. * P<0.05, vs. normal group. (C) mRNA levels of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSC control group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using quantitative polymerase chain reaction. (D) Protein expression of GAP-43 was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSCs group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using western blotting. The graph depicts the results of the densitometric quantification of the expression of GAP-43. 1, sham group; 2, GAP-43/BMSC group; 3, shGAP-43/BMSCE group; 4, PBS group; 5, GFP/BMSC group. (E) Hematoxylin and eosin staining revealed that pathological changes in the retina were markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups at day 3. Scale bar=50 µ m. * P<0.01 GAP-43/BMSC, vs. GFP/BMSC group, # P<0.05 shGAP-43/BMSC, vs. GFP/BMSC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; shGAP-43, transduced with shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; RGCs, retinal ganglion cells; TON, traumatic optic neuropathy; PBS, phosphate-buffered saline.

Article Snippet: The samples were then incubated with rabbit anti-rat nestin, NeuN and GAP-43 polyclonal antibodies (Wuhan Boster Biological Technology, Ltd.) at a dilution of 1:100 in PBS at 4°C overnight, washed twice with PBS and incubated with FITC- or Cy3-labeled rabbit anti-goat IgG (H+L; Wuhan Boster Biological Technology, Ltd.) at a dilution of 1:200 in PBS at room temperature for 1 h. Finally, images were captured using an inverted fluorescence microscope (Olympus Corp.).

Techniques: Injection, Staining, Labeling, Real-time Polymerase Chain Reaction, Expressing, Western Blot, shRNA, Transduction, Negative Control, Plasmid Preparation