Total cell proteins were prepared using IP lysate (P0013, Beyotime, China) mixed with protease and phosphatase inhibitors (HY-K0010 and HY-K0023, MCE, China). Cell lysates were centrifugated at 4℃ and their concentrations were determined using a BCA Protein Assay Kit (Pierce Biotechnology, USA). Next, the samples were subjected to separation through electrophoresis with 10–12% Tris SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with 5% skimmed milk at room temperature and primary antibodies at 4℃ overnight. Next, secondary horseradish peroxidase (HRP) antibodies were applied at room temperature. Finally, proteins were visualized using ECL (WB012, Share-Bio, Shanghai) detection regent and a Bio-Rad system. The antibodies used were directed against GFRA1 (ab84106, Abcam, 1:1000), GDNF (ab18956, Abcam, 1:1000), RET (14,556, Cell Signaling Technology, 1:1000), β-actin (30101ES50, Yeasen, China, 1:5000), mTOR (2983, CST, 1:1000), p-mTOR (5536, CST, 1:1000), S6K (2708, CST, 1:1000), p-S6K (9204, CST, 1:1000), BECN1 (3495, CST, 1:1000), LC3 (12,741, CST, 1:1000), P62 (8025, CST, 1:1000), cleaved caspase-3 (9661, CST, 1:1000) and its corresponding HRP-conjugated antibodies (anti-mouse, 115–035-003 and anti-rabbit, 111–035-003, Jackson ImmunoResearch, 1:10,000 for both).
Ab18956
Manufactured by Abcam
Sourced in United Kingdom
Ab18956 is a monoclonal antibody specifically recognizing the protein target. It is suitable for use in various immunoassay applications to detect the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab108319, by Abcam (5 mentions)
Ab8227, by Abcam (3 mentions)
Ab84106, by Abcam (2 mentions)
Ip lysate, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Share-Bio (1 mentions)
β actin, by Yeasen (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab7671, by Abcam (1 mentions)
Ab81321, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Ab46172, by Abcam (1 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Ab83760, by Abcam (1 mentions)
Rabbit anti β actin antibody, by Proteintech (1 mentions)
Vibratome, by Leica (1 mentions)
Tunel kit, by Roche (1 mentions)
Ab6199, by Abcam (1 mentions)
Ab9018, by Abcam (1 mentions)
Imager 600, by Cytiva (1 mentions)
15 protocols using ab18956
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of NRP1, Na/K-ATPase, GDNF
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After blocking by 5% skimmed milk, the samples were incubated with primary antibody (rabbit anti-NRP1, ab81321, Abcam, 1:250; mouse anti-alpha 1 Na/K-ATPase, ab7671, Abcam, 1:250; rabbit anti-GDNF, ab18956, Abcam, 1:100; mouse anti-β-actin, sc47778, Santa cruz, 1:1000; rabbit anti-Histone H3 antibody, BS1660, bioworld, 1:500) at 4°C overnight [57 (link)]. Then, the samples were incubated with IRdye secondary antibodies (goat anti-Rabbit, LI-COR, Odyssey, 1:1000; goat anti-Mouse, LI-COR, Odyssey, 1:1000) at room temperature for 2h. Finally, the protein bands were scanned by Odyssey imaging system (LI-COR, USA) and quantified with ImageJ software (National Institutes of Health, USA).
3
Western blot analysis of neurotrophic factors
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Sciatic nerves were rinsed in cold PBS and lysed on ice in radioimmunoprecipitation assay buffer containing a pro-tease inhibitor cocktail (Pulilai), and the resulting tissue lysates were mixed with sample buffer and boiled at 95°C for 5 minutes. Equal amounts of protein from each sample were subjected to 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Pulilai). The membranes were blocked in 5% nonfat dry milk at 4°C for 1 hour and incubated with rabbit anti-hepatocyte growth factor (HGF) antibody (1:1000; ab83760, Abcam, Cambridge, UK), rabbit anti-glial cell line-derived neurotrophic factor (GDNF) antibody (1:1000; ab18956, Abcam), rabbit anti-ciliary neurotrophic factor (CNTF) antibody (1:1000; ab46172, Abcam) or rabbit anti-β-actin antibody (1:1000; Proteintech, Chicago, IL, USA) at 4°C overnight. These were followed by the appropriate secondary antibody, donkey-anti-rabbit-HRP (1:5000, Pulilai) at room temperature for 1 hour. The membranes were developed using an enhanced chemiluminescence substrate (Thermo Fisher). Measurement of the protein band intensities was conducted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Immunohistochemical Analysis of HMGB1 and Apoptosis in Ischemic Brain
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Brains were isolated and fixed with 4% paraformaldehyde by transcardial perfusion and post-fixed in the same solution overnight at 4°C. Coronal brain sections (20 µm) from the ischemic core region were prepared using a vibratome (Leica, Solms, Germany), and immunological staining was performed using a previously described floating method [26] (link). Rabbit anti-HMGB1 (ab18956; Abcam, Cambridge, UK) antibody was used at a 1∶500 dilution. FITC-labelled anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody for anti-HMGB1.
The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assay was carried out with a commercial TUNEL kit (Roche, Switzerland) according to the manufacturer's instructions. Apoptotic cells in the rat heart after I/R in each group were counted separately using Image J software.
The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assay was carried out with a commercial TUNEL kit (Roche, Switzerland) according to the manufacturer's instructions. Apoptotic cells in the rat heart after I/R in each group were counted separately using Image J software.
5
Immunofluorescence Imaging of Brain Tissue
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For immunofluorescence imaging, 5 µm-thick paraffin-embedded brain sections were cut, deparaffinized and rinsed in PBS. Sections were incubated in PBS supplemented with 3% bovine serum albumin (BSA), 10% normal calf serum and 1% Triton X-100 for 1 h at room temperature to block nonspecific binding. Subsequently, the sections were incubated overnight at 4°C with mouse anti-neuronal nuclei (NeuN; 1:500; ab18956; Abcam, Cambridge, UK) and rabbit anti-glial fibrillary acidic protein (GFAP; 1:1,000; MA191029; Chemicon International, Inc., Temecula, CA, USA) monoclonal antibodies in PBS containing 1% Triton X-100. Following washing in PBS for 30 min (6 times for 5 min), the sections were incubated with fluorescein isothiocyanate-conjugated, affinity-purified anti-mouse IgG and anti-rabbit IgG (both 1:200; both Jackson Immuno-Research Laboratories, West Grove, PA, USA) for 2 h at room temperature. Following washing in PBS for 30 min, the sections were mounted with mounting medium and examined under a fluorescence microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan).
6
Molecular Profiling of Schwann Cells
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WB was performed using antibodies to VEGF (ab32152), nerve growth factor (NGF; ab6199), BDNF (ab108319), glial cell-derived neurotrophic factor (GDNF; ab18956), neural cell adhesion molecule 1 (NCAM1; ab9018), and growth associated protein 43 (GAP43; ab12274) purchased from Abcam, and to proliferating cell nuclear antigen (PCNA; #13110), protein kinase B (AKT; #4691), phosphorylated protein kinase B (p-AKT; #4060), CD31 (#77699), and GAPDH (#51332) purchased from Cell Signaling Technology.
Schwann cells and sciatic nerves were harvested from the experimental and control groups for WB. Briefly, Schwann cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Applygen) for protein extraction. Then, aliquots of 20 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), which were then incubated with antibodies as described above. Western blots were imaged using an Amersham Imager 600.
Schwann cells and sciatic nerves were harvested from the experimental and control groups for WB. Briefly, Schwann cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Applygen) for protein extraction. Then, aliquots of 20 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), which were then incubated with antibodies as described above. Western blots were imaged using an Amersham Imager 600.
7
Comprehensive Antibody Analysis Protocol
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Antibodies used are as follow; N-cadherin antibody (ab76011, Abcam, Cambridge, UK), proN-cadherin antibody (GTX101141, GeneTex, San Antonio, TX, USA), GDNF antibody (ab18956, Abcam, Cambridge, MA, USA), N-cadherin (phospho Y860) (ab119752, Abcam), p120-catenin (ab92514, Abcam), β-catenin (GTX22982, Genetex, San Antonio, TX, USA), β-catenin phosphorylated antibodies including phospho Y654 (ab24925, Abcam), phospho Y489 (ab138378, Abcam), phospho Y142 (ab27798, Abcam), Ser33/37/Thr41 (9561, CST, Danvers, MA, USA), nestin (ab22035, Abcam, Cambridge, MA, USA), rabbit anti-CD133 (ab19898, proteintech, America), and anti-Caveolin-1 (SAB4200216, Sigma, Aldrich, USA).
8
Western Blotting for Gene Expression Analysis
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Western blotting was used to detect the expression of GDNF and GFP genes in the
modified GDNF and vector groups at P2, P3, and P4 (Fig. 1B ). Following pretreatment, hAMSCs
were lysed in a radioimmunoprecipitation assay buffer (Sigma) with
phenylmethylsulfonyl fluoride at 4 °C and an equal amount of protein (10–30 μg)
was loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel
and transferred to a nitrocellulose membrane. The primary antibodies used were
anti-GDNF (1:500, ab18956, Abcam) and anti-β-actin (1:2000, ab8227, Abcam),
which were detected by chemiluminescence after incubation with horseradish
peroxidase (HRP)-conjugated secondary antibodies. Striatal proteins were
harvested from three randomly screened PD mice after 6-OHDA lesioning, following
which levels of tyrosine hydroxylase (TH) in the left and right striatum were
determined via Western blotting. Primary antibodies were diluted in blocking
solution as follows: rabbit anti-mouse TH, 1:500(ab75875, Abcam); rabbit
anti-mouse beta-actin, 1:2000(ab8227, Abcam). Moreover, Western blot was also
used to detect the expression of GFRa1 in these delivery vehicles. Primary
antibodies were diluted in blocking solution as follows: anti-GFRα1,
1:500(ab84106, Abcam) and anti-β-actin, 1:2000(ab8227, Abcam).
modified GDNF and vector groups at P2, P3, and P4 (
were lysed in a radioimmunoprecipitation assay buffer (Sigma) with
phenylmethylsulfonyl fluoride at 4 °C and an equal amount of protein (10–30 μg)
was loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel
and transferred to a nitrocellulose membrane. The primary antibodies used were
anti-GDNF (1:500, ab18956, Abcam) and anti-β-actin (1:2000, ab8227, Abcam),
which were detected by chemiluminescence after incubation with horseradish
peroxidase (HRP)-conjugated secondary antibodies. Striatal proteins were
harvested from three randomly screened PD mice after 6-OHDA lesioning, following
which levels of tyrosine hydroxylase (TH) in the left and right striatum were
determined via Western blotting. Primary antibodies were diluted in blocking
solution as follows: rabbit anti-mouse TH, 1:500(ab75875, Abcam); rabbit
anti-mouse beta-actin, 1:2000(ab8227, Abcam). Moreover, Western blot was also
used to detect the expression of GFRa1 in these delivery vehicles. Primary
antibodies were diluted in blocking solution as follows: anti-GFRα1,
1:500(ab84106, Abcam) and anti-β-actin, 1:2000(ab8227, Abcam).
9
Spinal Protein Expression Analysis
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After 12-week exercise, animals (n = 3 in each group) were killed and proteins were extracted from C5 to C7 spinal segments, which were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose and the blots were probed with anti-myelin basic protein (anti-MBP) rabbit polyclonal antibody (1:1,000, ab40390, Abcam), anti-TH rabbit polyclonal antibody (1:1,000, AB152, Millipore), anti-synaptophysin mouse polyclonal antibody (SY38; 1:500, ab8049, Abcam), anti-postsynaptic density-95 (anti-PSD-95) rabbit polyclonal antibody (1:500, 516900, Invitrogen), anti-insulin like growth factor-1 (anti-IGF-1) rabbit polyclonal antibody (1:2,000, ab9572, Abcam), anti-glial cell line-derived neurotrophic factor (anti-GDNF) rabbit polyclonal antibody (1:500, ab18956, Abcam), anti-BDNF (1:500, ab108319, Abcam), anti-β actin rabbit polyclonal antibody (1:5,000; ab8227, Abcam), anti-β tubulin rabbit polyclonal antibody (1:5,000; ab6046, Abcam). Peroxidase anti-rabbit IgG (1:5,000, ab6721, Abcam) and peroxidase anti-mouse IgG (1:10,000, Vector Laboratories) were used as secondary antibodies. Immunoreactivity was detected using an enhanced chemiluminescence (ECL) detection kit (1705061, Bio-Rad).
10
Immunohistochemical Analysis of BMP2 and GDNF Expression
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Immunohistochemistry was performed as previously described (Raible and Kruse, 2000 (link)). In brief, paraffin sections were first rehydrated, and then rehydrated sections were incubated with a 1:500 dilution of rabbit anti-human primary antibody against BMP2 (1:300, Abcam, ab14933) and GDNF (1:200, Abcam, ab18956), or IgG (1:1000, as a negative control, Abcam, ab172730) overnight at 4°C. The tissue sections were washed in PBS and then incubated with a 1:100 dilution of biotinylated secondary goat anti-rabbit IgG (1:1000, Jingmei BioTech, Shenzhen, China). After washing with PBS, tissue sections were incubated with an avidin-biotin complex and developed in 0.075% (w:v) 3,3 diaminobenzidine (DAB). After lightly counterstaining with hematoxylin, the sections were dehydrated.
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