Rat cortex and striatum tissue was homogenized in 150 μL of protein lysis buffer and protease inhibitors (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM EGTA; 2 mM sodium orthovanadate; 0.2 mM phenylmethylsulfonyl fluoride; 1 mM HEPES, pH 7.6; 1 μg/ml leupeptin; and 1 μg/ml aprotinin) using a Polytron homogenizer. Then the samples were sonicated and centrifuged at 14,000 g for 15 min at 4°C. The supernatant was collected and protein quantified using a bicinchoninic acid kit (Sigma). Proteins (30 μg) were separated on 4 to 20% gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in 5% BSA or milk in TBST (0.1% Tween 20 in 1× Tris-buffered saline) and incubated with primary antibodies overnight at 4°C. Primary antibodies used in this study included: COX1 (1:2000 Abcam); ND1 (1:2000, Abcam); NDUFS1 (1:2000, Abcam); cleaved Caspase 3 (1:1000, Cell Signaling, Danvers); Caspase 3 (1:1000, Santa Cruz); GAP-43 (1:1000, Cell Signaling) and GAPDH (1:10000, Fitzgerald). After incubation for 1hr at room temperature with secondary rabbit (1:2000, Abcam) or mouse (1:20000, Abcam) antibodies conjugated with horseradish peroxidase, membranes were detected by chemiluminescence. Densitometric analysis was performed using ImageJ [26 (link)].
Gap43
Manufactured by Cell Signaling Technology
Sourced in United States
GAP43 is a membrane-associated protein that is involved in the growth and guidance of axons during neural development. It is a well-established marker for axonal growth and synaptic plasticity.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Nf 200, by Cell Signaling Technology (2 mentions)
Neuronal nuclei (neun), by Cell Signaling Technology (2 mentions)
Bicinchoninic acid kit, by Merck Group (1 mentions)
Ndufs1, by Abcam (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Biosynth (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Eclipse ci l microscope, by Nikon (1 mentions)
Edta solution, by Solarbio (1 mentions)
Sa00013 1, by Proteintech (1 mentions)
Anti neurofilament 200 nf200, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Protein extraction kit, by Solarbio (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Af3242, by Affinity Biosciences (1 mentions)
Bs 6951r, by Bioss Antibodies (1 mentions)
P pi3k, by Affinity Biosciences (1 mentions)
Iba 1, by Cell Signaling Technology (1 mentions)
Txnip, by Cell Signaling Technology (1 mentions)
14 protocols using gap43
1
Quantifying Mitochondrial and Apoptotic Proteins in Rat Brain Regions
2
Immunofluorescence Staining of Spinal Cord and PC12 Cells
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Spinal cord tissues were fixed in a 10% neutral buffered formalin solution for 24 h. Following fixation, tissues underwent standard dehydration and embedding processes, were serially sectioned at 4 μm thickness, and the sections were subjected to antigen retrieval in EDTA solution (Solarbio, Beijing, China) using a microwave. PC12 cells were fixed using 4% paraformaldehyde for a duration of 20 min. Tissue and cell samples were permeabilized in Triton X-100 (Solarbio, Beijing, China) for 20 min, blocked with 3% BSA for 1 h at room temperature, and incubated overnight at 4°C with anti-neurofilament 200 (NF200) (1:200, 2,836, Cell Signalling Technology, Beverly, MA, United States) and growth-associated protein 43 (GAP43) (1:200, 8,945, Cell Signalling Technology, Beverly, MA, United States) primary antibodies. Subsequently, the samples were incubated with rabbit (1:200, Cat No: SA00013-1, Proteintech, Wuhan, China) or mouse (1:200, Cat No: SA00013-1, Proteintech, Wuhan, China) secondary antibodies conjugated with fluorochrome for 1 h, followed by re-staining of cell nuclei with DAPI for 1 h and sealing. Imaging was performed using a Nikon ECLIPSE CI-L microscope, and fluorescence intensity was quantitatively analyzed with Image J software.
3
Quantitative Protein Analysis in Spinal Cord
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The total protein from spinal cord tissues and cell samples was extracted using a protein extraction kit (Solarbio, Beijing, China), following the provided instructions, and the protein concentration was quantified using the BCA method. After gel preparation, the samples were loaded in equal amounts of total protein. The proteins was separated by 10% SDS-PAGE (80 V, 2 h, then 130 V, 1 h) and transferred to a PVDF membrane. The membrane was blocked with 5% milk for 1 h, followed by overnight incubation at 4°C with primary antibodies against NF200 (1:1000, 2,836, Cell Signalling Technology, Beverly, MA, United States), GAP43 (1:1000, 8,945, Cell Signalling Technology, Beverly, MA, United States), PI3K (1:1000, 4,292, Cell Signalling Technology, Beverly, MA, United States), p-PI3K (1:500, AF3242, Affinity, China), Akt (1:1000,bs-6951R, Bioss, Beijing, China), p-Akt (1:1000,bsm-60645R, Bioss, Beijing, China), and GAPDH (1:8000, 2,118, Cell Signalling Technology, Beverly, MA, United States). This was succeeded by incubation with HRP-conjugated secondary antibodies (1:10000, 7,074, 7,076, Cell Signalling Technology, Beverly, MA, United States) for 1 h at room temperature. Lastly, the membranes were visualized using ECL reagents and the Azure Biosystems NIR Fluorescence Imaging system, followed by quantitative grayscale analysis using Image J software.
4
Immunofluorescence Analysis of Spinal Cord
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In accordance with the manufacturer’s instructions, the expression of GAP-43, Iba-1, TXNIP, cleaved-Caspase-1 and NLRP3 expressions in four to six lumbar cord segments (L4-L6) were detected by immunofluorescence. Then, the samples were blocked with 1% BSA and 0.1% Triton X-100 for 60 min, GAP-43, Iba-1, TXNIP, cleaved-Caspase-1 and NLRP3 (1:500, Cell signaling, Beverly, MA, USA) antibodies were added. Secondary antibodies IgG (H + L) FITC (1:1000, 11–4011-85, ThermoFisher) were added and incubated in the dark for 2 h. The images were taken and quantified with the A Zeiss LSM 800 confocal laser scanning microscope at a magnification of 20×.
5
Protein Expression Analysis in Cells
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The cultured cells are washed 3 times with cold Phosphate Balanced Solution (PBS). Then the Radio-Immunoprecipitation Assay (RIPA) lysis buffer is added to the cells at 4°C for 10 minutes. The mixture is centrifuged at 12,000 rpm, 4°C for 10 minutes. 10% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is used to separate the equal amounts of the protein. When the proteins are transferred to the Polyvinylidene Fluoride (PVDF) membrane, the 5% BSA is used to bind the protein of the membrane for 1 h at room temperature. Subsequently, the membrane is incubated with primary antibodies which are the PTEN, NF-200, and GAP-43 (Cell Signal Technology, BSN, USA) at 4°C overnight. After washing 3 times with the PBS, a goat-anti-rabbit IgG-HRR secondary antibody (Thermo Pierce, MA, USA) is used for incubating the membrane at room temperature for 2 h. Protein levels are normalized to GAPDH by using a mouse monoclonal anti-GAPDH antibody (Thermo Pierce, MA, USA).
6
Histological Evaluation of Skin Regeneration
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Animals were sacrificed on postoperative day 12, the damaged skins were excised and fixed in 4% paraformaldehyde overnight. The fixed skins were embedded in paraffin, cut into slices for H&E and Masson’s staining according to universal protocols. For immunohistochemistry analysis, the skin slices were rehydrated, incubated in citrate antigen retrieval solution, blocked with 3% bovine serum albumin, stained using primary antibodies to alpha smooth muscle (α-SMA; Sabbiotech, USA), CD31 (Service Bio, Wuhan, China) and the neural regeneration marker growth associated protein 43 (gap43; Cell Signaling Technology, USA) and TUJ1 (Cell Signaling Technology, USA). After being incubated with appropriate secondary antibodies, the slides were reacted with DAB substrate solution (Service Bio, Wuhan, China) and counterstained with hematoxylin, mounted with resin. The images of the slides were captured by Nikon Eclipse Ti2-U microscope, and analyzed by ImageJ software.
7
Alpinetin Modulates Neuroinflammation Pathways
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Alpinetin (CAS. 36052‐37‐6, purity ≥98%) was purchased from Chemstrong Scientific Co., Ltd. Cy3‐labeled goat anti‐rat IgG, Alexa Fluor 555‐tagged donkey antirabbit/mouse IgG, Alexa Fluor 488‐tagged goat antirabbit/mouse IgG, horseradish peroxidase‐labeled secondary antibodies, antifluorescence quenching agent (containing DAPI), and CCK‐8 reagent were purchased from Beyotime. Lipopolysaccharide (LPS) was purchased from Merck. Anti‐COX‐2, anti‐Iba‐1, anti‐CD68, anti‐GFAP, and anti‐GAPDH antibodies were purchased from Abcam. Primary antibodies against iNOS, p‐JAK2, p‐STAT3, STAT3, β‐actin, Bcl‐xL, GFAP, NeuN, GAP43, and MAP2 were obtained from Cell Signaling Technology. Alexa Fluor 488 phalloidin was also from CST. The anti‐CD11b antibody was bought from Biolegend. The anti‐JAK2 antibody was provided by Affinity. WP1066 purchased from GlpBio. Transwell chamber was purchased from Corning (0.4 μm pores). Calcein AM/propidium iodide (PI) was bought from Solarbio. The JC‐1 MMP assay kit and the ROS fluorescence test kit were from Elabscience. The polymerase chain reaction (PCR) primers were synthesized by Servicebio while the kit for RNA extraction came from EZBioscience. The reagents for quantitative real‐time PCR (qPCR) were purchased from Vazyme.
8
GAP43 Protein Expression in SD Rats
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Que was purchased from Aladdin Chemical (Shanghai, China). All other reagents were obtained from Sigma-Aldrich Co (St Louis, MO, USA). The mAb GAP43 was purchased from Cell Signaling Technologies (Danvers, MA, USA). Male SD rats weighing 250–300 g were provided by the Animal Center of Jinzhou Medical University, and the animal protocols were approved by the Animal Protection and Use Committee of Jinzhou Medical University.
9
Protein Expression Analysis in Cells
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Cells were lysed in RIPA buffer supplemented with complete protease inhibitor. Proteins were separated by SDS-PAGE and transferred to HyBond-C-Extra nitrocellulose membranes. The following antibodies were used: CHGA (M0869, Dako), TH (Ab112, Abcam), N-MYC (Sc-791, Santa Cruz BioTechnology), SYP (M0776, Dako), GAP43 (8945, Cell Signaling), Actin (691001, MP Biomedicals), and SDHA (Ab14715, Abcam).
10
Plasmid Construction and Antibody Catalogue
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pEGFP-N1-Ngb wild-type (WT) and mutants (E53Q, E118Q, K119N, H64A, H64L, Y44D)19 (link) and pGenesil-1-shNgb (short hairpin Ngb)20 (link) were previously constructed. pEGFP-C1-Ngb WT and truncates (Δ1–6, Δ1–18, Δ1–35, Δ1–42, Δ106–151, Δ123–151, Δ128–151), pDEST26-CV-p38, and pDEST-Flag-NV-p38 were constructed in our laboratory. EST26-CV (C-terminal 137–238 Venus), pDEST26-CV-p38, pDEST-Flag-NV (N-terminal 1–157 Venus), pDEST-Flag-NV-p38, pDEST27-GST, and pDEST27-GST-p38 were kindly provided by Professor Haian Fu (Department of Pharmacology, School of Medicine, Emory University, USA). pDEST26-CV-Ngb and pDEST-Flag-NV-Ngb were previously constructed18 (link). p-FU-p38K53A mutant plasmid was constructed by PCR amplifying p38K53A fragment (with stop codon) using specific primers and pDEST-Flag-NV-p38 template and then cloning into p-FU-Venus plasmids at BamH1/BsrG1sites. All plasmids were confirmed by sequencing before use. Antibodies against Ngb, Flag (Sigma, Saint Louis, MO, USA), GAP43, GFAP, NeuN, NF200, p-p38, p38 (Cell Signaling Technology, Boston, USA), β-tubulin, green fluorescent protein (GFP), Cygb, GST, His (Santa Cruz Biotechnology, Santa Cruz, USA), and Tau-1 (Merk Millipore Ltd., Darmstadt, Germany) were purchased.
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