Antibodies against MMP-9 (ab228402), V5 (ab27671), Integrin β3 (ab119992), ICAM-1 (ab222736), VCAM-1 (ab134047), TRAF-6 (ab33915) and β-actin (ab8227) were purchased from Abcam. Anti- Integrin β5 (#3629), and NF-κB proteins (NF-κB Pathway Sampler Kit #9936) were purchased from Cell Signaling Technology. Pierce™ Protein A/G Plus Agarose was obtained from Invitrogen. LPS, rhodamine B isothiocyanate-dextran, and p-aminophenylmercuric acetate (APMA) were purchased from Sigma-Aldrich. Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems. MMP-9 neutralizing monoclonal antibody (IM09L) was from Sigma-Aldrich. NF-κB inhibitor, PDTC, and ROS scavenger, N-acetyl-L-cysteine (Nac), were obtained from MedChem Express (Shanghai, China). LipoJet™ reagent and GeneMute siRNA transfection reagent were purchased from SignaGen (Ijamsville, MD, USA).
Ab222736
Manufactured by Abcam
Sourced in United States, China
Ab222736 is a lab equipment product offered by Abcam. It is a device designed for scientific research purposes. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab134047, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab8227, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Rhodamine b isothiocyanate dextran, by Merck Group (1 mentions)
Lipojet reagent, by SignaGen (1 mentions)
Ab33915, by Abcam (1 mentions)
Ab27671, by Abcam (1 mentions)
N acetyl l cysteine nac, by MedChemExpress (1 mentions)
Genemute sirna transfection reagent, by SignaGen (1 mentions)
P aminophenylmercuric acetate apma, by Merck Group (1 mentions)
Ab228402, by Abcam (1 mentions)
Ripa protein extraction reagent, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ab53013, by Abcam (1 mentions)
Ab226977, by Abcam (1 mentions)
Ab39688, by Abcam (1 mentions)
Ab28472, by Abcam (1 mentions)
Ab126090, by Abcam (1 mentions)
11 protocols using ab222736
1
Antibody-based Exploration of Inflammatory Pathways
2
Protein Expression Analysis in HeLa Cells
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RIPA protein extraction reagent (Beyotime, Shanghai, China) with PMSF (Roche, Basel, Switzerland) was used to lysed HeLa cells. 10% SDS-PAGE was used to separate the protein and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). 5% non-fat milk was used to block the membranes for 2 h. The membranes were probed with anti-GSPT1 (Abcam, ab126090), anti-WNT3A (Abcam, ab28472), anti-ICAM1 (Abcam, ab222736), anti-Vimentin (Abcam, ab8069), anti-E-cadherin (Abcam, ab53013), anti-p-β-catenin (Abcam, ab11350), β-catenin (Abcam,ab32572), anti-c-myc (Abcam, ab39688), anti-cyclin D1 (Abcam, ab226977), anti-GAPDH (Abcam, ab181602) antibodies overnight at 4˚C. Subsequently, membranes were incubated with a HRP-conjugated secondary antibody (CST, #7074) for 1 h at 37˚C. Finally, the blots were visualized by ECL (Thermo Fisher Scientific) and detected using a ChemiDoc XRS imaging system. Band densities were analyzed using Image J software (National Institute of Health, Bethesda, MD, USA). Relative protein levels were determined by normalizing the densitometry value of the proteins of interest to that of GAPDH.
3
Aortic Adhesion Molecule Expression
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The expression levels and distribution of the AS marker proteins intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) in the aortic tissues of each group (n = 3) were measured using immunohistochemistry. After deparaffinization, the paraffin sections were incubated with 3% H2O2 for 20 min. Tissue antigen retrieval was performed at 121 °C for 20 min using citrate buffer (pH = 6.0). Then, 1% bovine serum albumin was added for 20 min. Rabbit anti-mouse ICAM (cat no. ab222736, Abcam) and VCAM (cat no. ab134047, Abcam) antibodies were added dropwise, and the tissues were incubated at 4 °C for 16 h. After washing away the unbound primary antibody, goat anti-rabbit IgG-HRP antibody (cat no. ab6721, Abcam) was added dropwise to the tissues and incubated at 37 °C for 1 h. 3,3’-Diaminobenzidine developer solution was used for color development. Hematoxylin was used to stain the nuclei. After dehydration with alcohol, the sections were mounted with neutral gum after the xylene became transparent. The results were photographed under an optical microscope (Leica). Statistical quantification of the positive densitometric values was performed using Image-Pro Plus 6.0.
4
Molecular Profiling of Aortic Proteins
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Total proteins of aortas were extracted by RIPA buffer (Thermo Fisher, USA) and then subjected to SDS PAGE. After transfer, the PVDF membrane was blocked with 5% blotting grade blocker (Bio-rad, Hercules, CA, USA). Then, primary antibodies including anti-NLRP3 (1:1000 dilution, ab270449, Abcam, China), anti-caspase-1 p20 (1:1000 dilution, ab138483, Abcam, China), anti-β-actin (1:5000 dilution, ab179467, Abcam, China), anti-ICAM-1 (1:1000 dilution, ab222736, Abcam, China), anti-VCAM-1 (1:2000 dilution, ab134047, Abcam, China) was added and incubated for overnight at 4°C. Next day, secondary antibodies were incubated for 1 h at room temperature. The ECL Substrate (Thermo Fisher, USA) was added to visualize the bands. The band intensity was quantitated and analyzed using ImageJ.
5
Protein Extraction and Western Blot Analysis
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Samples (lung tissue or lung vascular
endothelial cells) were dissolved in lysis buffer for lysis, centrifuged
at 12,000g for 5 min to collect their supernatants,
and analyzed for their protein concentration using a BCA kit. High-resolution
separation of protein samples (20 or 40 μg protein/well) was
obtained on a 12% SDS-PAGE gel. These proteins were then transferred
to nitrocellulose or PVDF membranes (Milipore). These membranes were
blocked for 10 min at room temperature using a commercial blocking
solution (PS108, Epizyme Biotech, Shanghai, China), washed three times
with TBST, and incubated with VE-cadherin (1:1000, ab205336, Abcam),
ZO-1 (1:1000, ab216880, Abcam), ICAM-1 (1:1000, ab222736, Abcam),
β-catenin (1:20000, Affinity), ITGAM (1:1000, #17800, CST),
ITGB2 (1:1000, #72607, CST), TSG101 (1:1000, ab125011, Abcam), CD9
(1:1000, A1703, Abclonal), Calnexin (1:1000, ab227310, Abcam), and
Syntenin (1:1000, ab19903, Abcam), overnight at 4 °C (approximately
12 h). These membranes were then washed three times with TBST and
incubated with a fluorescently labeled secondary antibody (antirabbit
or antimouse lgG, Cell Signaling Technology) for 60 min at room temperature.
After three washes, the prints were visualized using an imaging system
(Bio-Rad).
endothelial cells) were dissolved in lysis buffer for lysis, centrifuged
at 12,000g for 5 min to collect their supernatants,
and analyzed for their protein concentration using a BCA kit. High-resolution
separation of protein samples (20 or 40 μg protein/well) was
obtained on a 12% SDS-PAGE gel. These proteins were then transferred
to nitrocellulose or PVDF membranes (Milipore). These membranes were
blocked for 10 min at room temperature using a commercial blocking
solution (PS108, Epizyme Biotech, Shanghai, China), washed three times
with TBST, and incubated with VE-cadherin (1:1000, ab205336, Abcam),
ZO-1 (1:1000, ab216880, Abcam), ICAM-1 (1:1000, ab222736, Abcam),
β-catenin (1:20000, Affinity), ITGAM (1:1000, #17800, CST),
ITGB2 (1:1000, #72607, CST), TSG101 (1:1000, ab125011, Abcam), CD9
(1:1000, A1703, Abclonal), Calnexin (1:1000, ab227310, Abcam), and
Syntenin (1:1000, ab19903, Abcam), overnight at 4 °C (approximately
12 h). These membranes were then washed three times with TBST and
incubated with a fluorescently labeled secondary antibody (antirabbit
or antimouse lgG, Cell Signaling Technology) for 60 min at room temperature.
After three washes, the prints were visualized using an imaging system
(Bio-Rad).
6
Western Blot Analysis of Vascular Markers
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Lysates from cells or tissues were prepared with RIPA lysis. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat dairy milk and incubated with primary antibodies against anti-VCAM-1 (1 : 1000 dilution, ab134047, Abcam), anti-ZFP36 (1 : 500 dilution, sc-374305, Santa Cruz), anti-Bcl-2 (1 : 1000 dilution, sc-7382, Santa Cruz), anti-Bax (1 : 1000 dilution, sc-7480, Santa Cruz), anti-cleaved caspase-3 (1 : 1000 dilution, ab49822, Abcam), anti-pro-caspase-3 (1 : 1000 dilution, ab32499, Abcam), anti-SM22α (1 : 1000 dilution, ab14106, Abcam), anti-ICAM-1 (1 : 1000 dilution, ab222736, Abcam), anti-IGFBP7 (1 : 500 dilution, DF7131, Affinity Biosciences), and anti-TNC (1 : 500 dilution, DF8051, Affinity Biosciences) at 4°C overnight. GAPDH (1 : 1000 dilution, ab181602, Abcam) was used as an internal control. This was followed by incubation with an IRDye800®-conjugated secondary antibody (1 : 20000 dilution, Rockland) for 1 h at room temperature and subsequent scanning with the Odyssey Infrared Imaging System (LI-COR Biosciences). The integrated intensity for each detected band was determined using Odyssey Imager software. Data are presented as mean ± SD from at least three independent experiments.
7
Western Blot Protein Analysis
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Protein was extracted from cultured cells using radioimmunoprecipitation assay (RIPA) buffer containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 1 mM; Beyotime, Jiangsu, China). Protein lysates were electrophoresed on 10 % SDS polyacrylamide gels, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA), blocked with 5 % skimmed milk for 1 h, and then incubated with primary antibodies including VEGF (1:1,000, ab52917), MMP-2 (1:1,000, ab92536), ICAM1 (1:1,000, ab222736) (all from Abcam, USA), α-SMA (1:1,000, #19245), or GAPDH (1:1,000, #2118 (both from CST, USA) at 4 °C overnight. After washing with TBST buffer, the membranes were treated with the secondary antibodies for 1 h at room temperature. Finally, the bands were demonstrated using an Enhanced Chemiluminescence (ECL) Plus Kit (Millipore) and the gray values were calculated through ImageJ software.
8
Protein Expression Analysis in OS Cells
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Total protein was isolated from OS cells. The concentration of the protein was calculated with BCA Kit (Beyotime, Shanghai, China). An equal amount of the protein was separated with 12% SDS-PAGE. Afterward, the proteins were moved onto Polyvinylidene Fluoride membranes (Millipore, Burlington, MA; USA). Then the membranes were sealed with 5% skimmed milk and incubated with primary antibodies, anti-ICAM-1 (ab222736, 56 kDa, 1: 1000, Abcam, Cambridge, MA, USA), anti-VCAM-1 (ab134047, 81 kDa, 1: 2000, Abcam), anti-CGN (ab117796, 136 kDa, 1: 2000, Abcam), anti-Bcl-2 (ab32124, 26 kDa, 1: 1000, Abcam), anti-BAX (ab32503, 21 kDa, 1: 1000, Abcam), anticleaved Caspase3 (ab32042, 17 kDa, 1: 500, Abcam), anticleaved- Caspase9 (ab2324, 37 kDa, 1: 1000, Abcam), and anti-β- actin (ab8227, 42 kDa, 1: 1000, USA) overnight at 4°C in shade. Lately, the membranes were washed with PBS 3 times and incubated with goat-anti-rabbit secondary antibodies (ab6721, 1: 2000, Abcam) for 2 h at 37°C. The protein in each band was determined with ECL solution (Millipore) in the shade. The protein levels were analyzed with ImageJ software 1.6.
9
Immunofluorescence Assay for Cell Markers
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Cells were sequentially fixed, permeabilized, and blocked. The primary antibodies applied included anti-CCL21 (#AF366, R&D), ICAM1 (#Ab222736, Abcam), and CD11α (#26703, CST) overnight. Fluorophore-conjugated secondary antibody (#A-11008, #A-11004, #A48255, #32733, Invitrogen) was then applied for 1 hour. After washing the cells with phosphate-buffered saline (PBS), they were stained with 4,6-diamidino-2-phenylindole (DAPI) (#C1005, Beyotime). The resulting cells were finally captured under a Zeiss LSM 710 confocal microscope.
10
Myocardial Protein Extraction and Analysis
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Total proteins from myocardial tissue were extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (cat. No. R0020; Solarbio, Beijing, China). Protein was quantified using the BCA protein assay kit (cat. No. P0012; Beyotime, Beijing, China). Proteins (40 ug) were separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose filter membranes (Millipore, Billerca, MA, USA) for 100 min at 300 mA. Then, the membranes were blocked for 1.5 h at room temperature with 5% skimmed milk or 3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween (TBS-T). The membranes were incubated with primary antibodies against ETBR (cat. No. ab262700, 1:1,000; Abcam, Fremont, CA, USA), ICAM-1 (cat. No. ab222736, 1:1,000; Abcam, Fremont, CA, USA), and GAPDH (cat. No. ab37168, 1:1,000; Abcam, Fremont, CA, USA) at 4 °C overnight and with a secondary antibody (cat. No. SA00001-2, 1:5,000; Proteintech, Rosemont, IL, USA) at room temperature for 1 h. The proteins on the membranes were visualized using an enhanced chemiluminescence (ECL) kit (cat. No. PK10003; Proteintech, Rosemont, IL, USA). The exposed protein bands were converted to data using Image software (Bio-Rad, Hercules, CA, USA) to obtain grayscale values for result analysis.
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