Western blotting in VSMCs and coronary tissues was performed as previously reported [29 (link)]. The following antibodies were used: anti-Kv1.2, anti-Kv1.5, anti-RAGE, and anti-β-actin (all from Abcam, U.K.).
Anti rage
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-RAGE is a primary antibody that detects the receptor for advanced glycation end products (RAGE). RAGE is a transmembrane protein that plays a role in various cellular processes. This antibody can be used in applications such as Western blotting and immunohistochemistry to study the expression and localization of RAGE in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (4 mentions)
Anti hmgb1, by Abcam (3 mentions)
Hrp conjugated secondary antibody, by Beyotime (3 mentions)
Anti smad7, by Abcam (3 mentions)
Anti p38, by Abcam (3 mentions)
Anti p p38, by Abcam (3 mentions)
Anti smad3, by Abcam (3 mentions)
Beyoecl plus, by Beyotime (3 mentions)
Anti p smad2, by Abcam (3 mentions)
Anti smad2, by Abcam (3 mentions)
Anti p smad3, by Abcam (3 mentions)
Anti β actin, by Abcam (2 mentions)
Anti inos, by Abcam (2 mentions)
Anti tlr4, by Abcam (2 mentions)
Anti gfap, by Abcam (2 mentions)
Ham s f12 medium, by Merck Group (2 mentions)
Ammonium pyrrolidinedithiocarbamate pdtc, by Beyotime (2 mentions)
Anti kv1, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti total p38, by Cell Signaling Technology (1 mentions)
27 protocols using anti rage
1
Western Blot Analysis of VSMC and Cardiac Tissues
2
Western Blot Analysis of Cardiac Proteins
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Western blots to analyse protein expression were conducted using the standard protocol as described previously 17 , 18 . Primary antibodies used were anti‐L‐type Ca2+ channel (Abcam, Cambridge, UK), anti‐ryanodine receptor (Abcam), anti‐SERCA2a (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐NCX (Cell Signaling, Danvers, MA, USA), anti‐phospho‐PLB (Millipore, Bellerica, MA, USA), anti‐total‐PLB (Millipore), anti‐PMCA1 (Abcam), anti‐PMCA4 (Abcam), anti‐RAGE (Abcam), anti‐phospho‐p38 (Cell Signaling), anti‐total‐p38 (Cell Signaling), anti‐iNOS (Abcam), anti‐α‐tubulin (Calbiochem) and anti‐GAPDH (Santa Cruz). For visualization, we used HRP‐conjugated secondary antibodies, which were obtained from either Cell Signaling or Dako.
3
Immunohistochemical Analysis of Rat Spinal Cord Tissue
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Animals were anesthetized with sodium pentobarbital (60 mg/kg body weight) and
perfused with phosphate-buffered saline (PBS) followed by fresh 4%
paraformaldehyde. L3-5 SDHs were collected from rats, fixed in 4%
paraformaldehyde overnight and cryopreserved in 30% sucrose at 4°C overnight.
Tissues were mounted and sectioned on a cryostat at a thickness of 12 µm. Tissue
sections were permeabilized with 0.3% Triton X-100 (Amresco, Solon, USA) in PBS
for 15 min, followed by antigen retrieval with Quick Antigen Retrieval Solution
for Frozen Sections (Beyotime, Jiangsu, China). Then, the sections were
incubated with 3% BSA for 1 h at room temperature and then with primary
antibodies overnight at 4°C. The following primary antibodies were used:
anti-glial fibrillary acidic protein (GFAP; Abcam, Cambridge, UK), anti-ionized
calcium binding adaptor molecule 1 (IBA1; Abcam, Cambridge, UK), anti-NeuN
(Abcam, Cambridge, UK), anti-p-NF-κB (Abcam, Cambridge, UK) and anti-RAGE
(Abcam, Cambridge, UK). The tissue sections were washed three times and
incubated with the appropriate secondary antibodies for 1 h at room temperature.
After the slides were washed in PBS, coverslips were applied with mounting
medium with DAPI (ZSGB-BIO, Beijing, China). The sections were examined on an
Olympus fluorescence microscope (Olympus, Tokyo, Japan).
perfused with phosphate-buffered saline (PBS) followed by fresh 4%
paraformaldehyde. L3-5 SDHs were collected from rats, fixed in 4%
paraformaldehyde overnight and cryopreserved in 30% sucrose at 4°C overnight.
Tissues were mounted and sectioned on a cryostat at a thickness of 12 µm. Tissue
sections were permeabilized with 0.3% Triton X-100 (Amresco, Solon, USA) in PBS
for 15 min, followed by antigen retrieval with Quick Antigen Retrieval Solution
for Frozen Sections (Beyotime, Jiangsu, China). Then, the sections were
incubated with 3% BSA for 1 h at room temperature and then with primary
antibodies overnight at 4°C. The following primary antibodies were used:
anti-glial fibrillary acidic protein (GFAP; Abcam, Cambridge, UK), anti-ionized
calcium binding adaptor molecule 1 (IBA1; Abcam, Cambridge, UK), anti-NeuN
(Abcam, Cambridge, UK), anti-p-NF-κB (Abcam, Cambridge, UK) and anti-RAGE
(Abcam, Cambridge, UK). The tissue sections were washed three times and
incubated with the appropriate secondary antibodies for 1 h at room temperature.
After the slides were washed in PBS, coverslips were applied with mounting
medium with DAPI (ZSGB-BIO, Beijing, China). The sections were examined on an
Olympus fluorescence microscope (Olympus, Tokyo, Japan).
4
Amygdala and Hippocampus Protein Analysis
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Six amygdala and hippocampus were preserved after weighing and were homogenized with radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors. The homogenates were centrifuged at 12,000 rpm for 15 min at 4°C. Western blotting was carried out using the standard method as previously described (Zhu et al., 2018 (link)). The primary antibodies used were as follows: anti-Glo-1, 1:1,000 (Abcam, UK); anti-RAGE, 1:1,000 (Abcam, UK); anti-Nrf2, 1.5:2,000 (Abcam, UK); anti-γ-GCS 1:1,000 (Bioworld Technology Inc., USA); and β-actin 1:1,000 (Bioworld Technology Inc., USA).
5
Quantitative Protein Expression Analysis
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Extraction of the total protein was performed from the frozen tissues with the aid of lysis buffer (Solarbio, China). BCA assay was used to measure the protein concentration (Sangon, China) according to the manufacturers’ protocol. Separation of total protein samples was executed using 10% SDS polyacrylamide gel electrophoresis, and then transferred onto the PVDF (Millipore, USA). Later, the transmembrane was subjected to blocking using a blocking buffer made of ‘5% nonfat milk, 0.1% Tween 20’. Later, the membranes were probed with anti-HMGB1 (1:1000 dilution, Abcam, USA), anti-RAGE (1:2000 dilution, Abcam, USA) primary antibodies and incubated overnight at 4 °C, respectively. Next, the membranes were washed 3 times using PBS. Then, the membranes were subjected to incubation for two hours at room temperature with secondary antibodies (1:2000 dilution, Odyssey CIX, USA). The blots were observed using Infrared Laser Scanning Imaging System (Odyssey CIX, USA).
6
Investigating HMGB1-NLRP3 Inflammasome Interactions
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Recombinant human high mobility group box 1 (HMGB1), anti-NLRP3, and anti-IgG antibodies were purchased from R&D Systems, Inc. (Minneapolis, MN). NLRP3-inflammasome inhibitor, MCC-950 was purchased from Invivogen (San Diego, CA). Anti-IL-1β, anti-TLR2, anti-TLR4, anti-RAGE, and anti-caspase-1 antibodies were from Abcam (Cambridge, MA). β-Actin antibody was purchased from Santa Cruz Biotechnology Inc. (Beverly, MA), and anti-ASC antibody from Adipogen (San Diego, CA).
7
Western Blot Analysis of Signaling Proteins
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Primary human arterial endothelial cells and PASMC were lysed in RIPA buffer (Sigma-Aldrich) and 10 μg of protein were run on a 10% SDS polyacrylamide gel, followed by electrotransfer to an Amersham Hybond P 0.45 PVDF Blotting Membrane (GE Healthcare, Wien, Austria). After blocking with 5% non-fat dry milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20), the membrane was incubated overnight at 4°C with one of the following antibodies: anti-p38, anti–phospho-p38 (T180/Y182), anti-c-Jun; anti-phospho c-Jun (S73), anti-phospho Erk1/2 (T202/Y204), anti-Erk1/2, anti-phospho c-Fos (S32) all from Cell Signaling and anti-c-Fos (Novus Biologicals, Cambridge, UK) were used at dilution of 1:1000; and anti-TLR4 (Abcam), anti-RAGE (1:500; Abcam) and rabbit anti–alpha-tubulin (1:5000; Cell Signaling, Boston, MA, USA). After washing, the membranes were incubated with horseradish-peroxidase–labelled secondary antibodies (1:5000, anti-rabbit-HRP, Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and proteins detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare). Antibodies were removed by incubating the membrane for 15 min. with stripping buffer (RestoreTM PLUS Western Blot Stripping Buffer; Thermo Scientific). Densitometric analysis was performed with ImageJ (National Institutes of Health, USA).
8
Molecular Biomarkers in Alzheimer's Mice
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Ipsilateral hippocampi to the injection site were dissected from frozen excised brains of vehicle-, Aβ-, and pentamidine-treated mice and lysed with ice-cold hypotonic lysis buffer (Tris/HCl pH 7.5 50 mM; NaCl 150 mM; EDTA 1 mM; Triton X-100 1%) supplemented with the proper protease inhibitor cocktail (Roche, Milan, Italy) and incubated on ice for additional 15 min. Total protein extracts were obtained by centrifugation at 13,000 g for 15 min at 4°C. Samples were subjected to SDS-polyacrylamide gel electrophoresis and proteins were transferred onto nitrocellulose membrane and incubated with one of the following antibodies: anti-GFAP (1 : 50000); anti-iNOS (1 : 200); anti-COX-2 (1 : 1000), anti-phospho(p)-p38 MAPK (1 : 400), anti-RAGE (1 : 1000), and anti-β-actin (1 : 2000) (all from Abcam). After wash in TBS 1X with 0.1% Tween 20, the membrane was incubated for 2 h at room temperature with the appropriate secondary HRP-conjugated antibodies anti-mouse (1 : 1000, Abcam) or anti-rabbit (1 : 1000, Abcam). Lastly, the membrane was exposed to the enhanced chemiluminescence substrate (ECL, Invitrogen, Milan, Italy), the immunoreactive bands were revealed through a Versadoc (Bio-Rad Laboratories, Milan, Italy) and the digital images were analyzed with the Quantity One Software (Bio-Rad Laboratories).
9
Quantitative Western Blot Analysis
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Tissue samples were separated by sodium dodecylsulfate – polyacrylamid gelelectrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Cat# 05317-10EA, Immobilon-FL) following standard protocols. After blocking with Odyssey® Blocking buffer, membranes were incubated overnight at 4 °C with the indicated primary antibodies: anti-RAGE (#ab3611, Abcam), anti-OGT (#24083, Cell Signaling) and anti-phospho histone H2A.X (#2577, Cell Signaling) followed by IRDye 800 CW secondary antibodies (P/N 925–32211, LI-COR). Bound complexes were detected using the LI-COR Odyssey® Fc imaging system (LI-COR, Germany). For quantification, the intensities of the total protein were normalised to REVERT™ total protein (LI-COR) signal.
10
Glycan-Specific Antibody Characterization
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The following antibodies were used: Anti-Paucimannose (serum-free undiluted cell culture supernatant; clone: Mannitou, gift from B. Schmitz, Bonn, Germany); anti high-mannose (serum-free undiluted cell culture supernatant; clone 492, gift from B. Schmitz, Bonn, Germany); anti-polySia (1:10,000 dilution, clone 735 gift from R.Gerady-Schahn, Hannover Germany, IgG), anti-NCAM (1:1000 dilution clone 5B8 IgG Hybridoma Bank); anti-CML (1:10,000 dilution; clone CML56 abcam); anti-O-GlcNAc (1:10,000 dilution; clone CTD110.6, Cell Signaling); anti-Synapsin-1 (1:5000 dilution; polyclonal 64581 Abcam); anti-RAGE (1:1000 dilution; polyclonal 3611 Abcam).
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