The bEND5 cells under different conditions were treated with various doses of ODNs or equal doses of the scramble control ODNs. RNA samples were evaluated by RT-PCR and protein samples were analyzed by western blotting to determine nuclear levels of P-65 and expression of ICAM-1 and/or VCAM-1. β-Actin was used as a protein loading control marker for whole cell lysates and TATA binding protein (TBP) was used as a protein loading control marker for nuclear protein. The following antibodies were used in western blotting procedure: 1: 2,000 (dilution) β-Actin antibody, 1:2000 NF-κB p65 antibody, 1;1000 phospho-NF-κB p65 (Ser536) antibody (Cell Signaling Technology, Danvers, MA), 1:1,000 ICAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), 1:2,000 VCAM-1 antibody and 1:1,000 TBP antibody (Abcam, Cambridge, MA). mRNA expression was measured using the following forward and reverse qPCR primers: VCAM-1: 5′-AAG ACT GAA GTT GGC TCA CAA-3′ and 5′-GGA GTT CGG GCG AAA AAT AG-3′; ICAM-1: 5′-GGG AAT GTC ACC AGG AAT GT-3′ and 5′-CAG TAC TGG CAC CAG AAT GA-3′; β-actin: 5′-GGC TGT ATT CCC CTC CAT CG-3′ and 5′-CCA GTT GGT AAC AAT GCC ATG T-3′. The mRNA expression of β-actin was used as an internal control. All the primers were purchased from Integrated DNA Technologies Inc.
Vcam 1 antibody
Manufactured by Abcam
Sourced in United States
The VCAM-1 antibody is a laboratory reagent used in research applications. It specifically binds to the vascular cell adhesion molecule-1 (VCAM-1), which is a cell surface protein involved in cell-cell adhesion. The antibody can be used to detect and study the expression and localization of VCAM-1 in various biological samples.
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Lab products found in correlation
Icam 1 antibody, by Abcam (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65 ser536 antibody, by Cell Signaling Technology (1 mentions)
Icam 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Leica microscope, by Leica camera (1 mentions)
Rapamycin, by Merck Group (1 mentions)
C fos antibody, by Abcam (1 mentions)
Total p38, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Gata6, by Cell Signaling Technology (1 mentions)
Total stat3, by Cell Signaling Technology (1 mentions)
Phospho shp 2, by Cell Signaling Technology (1 mentions)
Histone h3 antibody, by Proteintech (1 mentions)
Phospho stat3 y705, by Cell Signaling Technology (1 mentions)
Stattic, by Selleck Chemicals (1 mentions)
Fr180204, by Selleck Chemicals (1 mentions)
Hydroxychloroquine sulfate cq, by Merck Group (1 mentions)
9 protocols using vcam 1 antibody
1
Evaluating NF-κB Pathway Regulation in bEND5 Cells
2
Immunohistochemical Analysis of VCAM-1 in Kidney
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For immunohistochemistry staining, the kidney sections were dewaxed, rehydrated, and antigen retrieved by autoclave in Tris/ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.0). The endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide for 10 min and washing with PBS 3 times. Non-specific binding was blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature. Tissue sections were, respectively, incubated with rabbit anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:200, Abcam, ab134047) overnight at 4°C. After washing 3 times with PBS, the tissue sections were incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit secondary antibody (Zhongshan Gloden Bridge Biotechnology, Beijing, China) for 20 min at room temperature, then stained with a 3,3’-diaminobenzidine (DAB) kit (Zhongshan Gloden Bridge Biotechnology, Beijing, China), and visualized under Leica microscope (Germany). Image Pro Plus (IPP) version 6.0 was used to assess the location and intensity of the positive area.
3
ICAM-1 and VCAM-1 Quantification Protocol
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The blood samples were centrifuged at 4oC for 10 min at 3000 rpm/min, and the plasma aliquots were separated and transferred into centrifuge tubes according to the instructions of the Kit (ICAM-1 antibody [Abcam Inc., Cambridge, MA, USA, ab174445] [32 (link)] and VCAM-1 antibody [Abcam Inc., Cambridge, MA, USA, ab46118] [33 (link)]). The serum samples to be tested were diluted, added into enzyme coated plates, and incubated at 37oC for 30 min. Each well was added with 50 μL of ELISA reactive solution, and incubated at 37oC for 30 min. Subsequently, each well was added with 50 μL of chromogenic agent A and 50 μL of chromogenic agent B, mixed by shaking for 30 s, and colored at 37oC in the dark for 15 min. The optical density (OD) values of each well were measured at a wavelength of 450 nm. Standard curves were then drawn with OD value as abscissa and concentration as vertical coordinate. The concentrations were 25, 50, 100, 200 and 400 ng/L.
4
Cellular Signaling Pathway Modulation
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tBHQ, rapamycin, and hydroxychloroquine sulfate (CQ) were obtained from Sigma Aldrich (Sigma Aldrich, MO). TRIzol™ Reagent, RNAiMAX, and Rapid Gold BCA Protein Assay Kit were provided by Thermo Fisher (Thermo Fisher Scientific, MA). STAT3 inhibitor Stattic, SHP2 inhibitor SHP099, p38 inhibitor SB202190, and ERK1/2 inhibitor FR180204 were bought from Selleck (Selleck Chemicals, TX). Recombined TNFα (both human and mouse) was bought from R&D Systems (R&D Systems, MN). tBHQ was dissolved in ethanol; hydroxychloroquine sulfate was dissolved in sterilized water, and other compounds were dissolved in dimethyl sulfoxide (DMSO). VCAM-1 antibody, c-Fos antibody, and p38 activator U46619 were purchased from Abcam (Cambridge, UK); histone H3 antibody was obtained from Protein-tech (Protein-tech, IL). GATA6, ATG7, phospho-p38, total-p38, phospho-Y705-STAT3, total-STAT3, NFκB (p65), SP-1, c-Jun, Nrf2, phospho-SHP2, and total SHP2 antibodies were purchased from Cell Signaling Technology, Inc. (Cell Signaling Technology, MA); LC3 and peroxidase conjugated β-actin antibodies were obtained from Sigma Aldrich (Sigma Aldrich, MO); horseradish peroxidase conjugated secondary antibodies was purchased from Jackson ImmunoResearch Inc. (Jackson ImmunoResearch, PA).
5
Aortic Root Histology and Immunohistochemistry
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Histological and immunohistochemical analyses were performed on frozen sections of the aortic root. The sections (at 5-µm intervals) were stained with Oil Red O to detect lipid deposition. Moreover, sections were incubated with monocyte chemoattractant protein-1 (MCP-1) antibody (BD Pharmingen), monocyte/macrophage marker 2 (MOMA2) antibody (Bio-Rad), or anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (Abcam) followed by the avidin–biotin complex technique and stained with a Vector Red Substrate Kit (Vector Laboratories, Inc.). Each section was counterstained with hematoxylin. The ratio of positive area to plaque area was calculated in three valve lesions in the aortic root and used for comparison
32) .
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Histological and immunohistochemical analyses were performed on frozen sections of the aortic root. The sections (at 5-µm intervals) were stained with Oil Red O to detect lipid deposition. Moreover, sections were incubated with monocyte chemoattractant protein-1 (MCP-1) antibody (BD Pharmingen), monocyte/macrophage marker 2 (MOMA2) antibody (Bio-Rad), or anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (Abcam) followed by the avidin–biotin complex technique and stained with a Vector Red Substrate Kit (Vector Laboratories, Inc.). Each section was counterstained with hematoxylin. The ratio of positive area to plaque area was calculated in three valve lesions in the aortic root and used for comparison
32) .
6
VCAM1+ SMC Isolation from Ligated Arteries
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The left carotid artery of C57BL/6 mice was completely ligated with 6-0 silk sutures just below the bifurcation point (n = 3). To obtain enough VCAM1+ SMCs for FACS sorting, the arteries above the ligation site were harvested on day 7 and digested to isolate the cells. VCAM1 antibody (catalog ab223982, Abcam) and VE-cadherin antibody (catalog 562242, BD Biosciences) were used to stain the cells, and a FACS system (Aria SORP, BD Biosciences) was used to sort VCAM1+VE-cadherin– SMCs and VCAM1–VE-cadherin– SMCs from normal and ligated arteries. The sorted cell number was counted by the FACS system in each group.
7
Immunofluorescence Assay for VCAM-1 Expression
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Immunofluorescence assay was carried out as following: briefly, HAVSMCs that were exposed to 150 μg/ml of ox-LDL gradually underwent paraformaldehyde and blockage. Then, the cells were reacted with VCAM-1 antibody (1:50, Abcam) overnight at 4°C, followed by incubation of the FITC–conjugated secondary antibody for 1 h. After washing with PBS, cells were observed by fluorescence microscopy.
8
Immunofluorescence Staining of VCAM-1 and α-SMA
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Tissue slides were stained for VCAM‐1 expression using VCAM‐1 antibody (dilution 1:200) purchased from Abcam (Cambridge, MA, USA) (ab134047). At the same time, slides were co‐localized with antibody to α‐smooth muscle actin (α‐SMA) (dilution 1:900, AA132; Beyotime). To detect specific antibody binding, secondary fluorescein‐conjugated antibodies (dilution 1:1000, ab150113 and ab150077; Abcam) were used, and cell nuclei were stained with DAPI (4',6‐diamidino‐2‐phenylindole; C1002; Beyotime). Digital images were recorded using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Density values of positive areas were quantified for statistical analysis using imagej , version 1.45.
9
Protein Expression Analysis of Endothelial Cells
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Micro-vascular endothelial cells were treated with ice-cold lysis buffer containing 10 mmol/l PMSF (pH, 7.5). The lysates were then subjected to SDS-PAGE and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. PVDF membranes were incubated overnight at 4ºC with one of the following primary antibodies, including rabbit anti-mouse ELAM-1 antibody, VCAM-1 antibody, ICAM-1 antibody, IKBα antibody, p-IKBα antibody, and GAPDH antibody (all antibodies were purchased from Abcam Biotechnology, Cambridge, Massachusetts, USA). Subsequently, the PVDF membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Beyotime Biotechnology Inc.), at room temperature for 1 hour. Bound antibodies in the above PVDF membranes were developed with chemiluminescent substrate (ECL, Beyotime Biotechnology Inc.), according to the instruction of manufacturer. The immunoreactive bands were imaged and scanned with gel imaging system (Tannon-4200, Tannon, Shang, China).
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