Tissue pieces were prepared by adding protease and phosphatase inhibitor cocktail in RIPA buffer and centrifuging for 10 minutes at 12000x rpm to remove debris. Western blot was performed as previously described (22 (link)–24 (link)). From the lysates, protein concentrations were determined by the Lowry protein assay (500–0113, 500–0114, 500–0115; Bio-Rad). Equal amounts of protein were loaded into each lane of a 4–12% Tris gel (BioRad) and subjected to electrophoresis. After blotting, nitrocellulose-membranes (BioRad) were blocked for 1h (milk powder 5% in TBS/Tween 0.1–0.2%) and incubated with primary antibodies (ICAM-1, R&D Systems, AF796; E-Selectin, Santa Cruz, sc-137054; G10 anti-Reelin, made in-house; Reelin, Millipore, MAB5366; ApoER2, made in-house; GAPDH, Sigma-Aldrich, G8795; α-Tubulin, Millipore, CP08). Binding of secondary HRP-antibodies were visualized by ECL or ECL plus chemiluminescent (Amersham). After densitometric analyses with ImageJ, optical density values were expressed as arbitrary units and normalized for protein loading, as described in the figure legends.
E selectin
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
E-selectin is a cell adhesion molecule that mediates the initial attachment of leukocytes to the vascular endothelium during the inflammatory response. It plays a crucial role in the recruitment and migration of immune cells to sites of inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Vcam 1, by Santa Cruz Biotechnology (12 mentions)
Icam 1, by Santa Cruz Biotechnology (11 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Gapdh, by Merck Group (3 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (3 mentions)
Lowry protein assay, by Bio-Rad (3 mentions)
Mab5366, by Merck Group (3 mentions)
Ecl plus chemiluminescent, by Cytiva (3 mentions)
Icam 1, by R&D Systems (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Nf κb p65, by Santa Cruz Biotechnology (3 mentions)
P selectin, by Santa Cruz Biotechnology (3 mentions)
Interleukin 6 (il 6), by Santa Cruz Biotechnology (3 mentions)
α tubulin, by Merck Group (2 mentions)
Poly l lysine coated slide, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Santa Cruz Biotechnology (2 mentions)
Il 10, by Santa Cruz Biotechnology (2 mentions)
P iκbα, by Santa Cruz Biotechnology (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
24 protocols using e selectin
1
Protein Extraction and Western Blot Analysis
2
Pulmonary Endothelial Cell Culture Protocol
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Human pulmonary artery endothelial cells (HPAECs) were obtained from Lonza (Allendale, NJ) and used at passages 5–8. All experiments were performed in EGM growth medium (Lonza) containing 2% fetal bovine serum (FBS) unless otherwise specified. Texas Red–conjugated phalloidin and Alexa Fluor 488–labeled secondary antibodies were purchased form Molecular Probes (Eugene, OR). TRAP6 was obtained from AnaSpec (San Jose, CA). 8CPT was obtained from Calbiochem (La Jolla, CA). GGTI-298 and thrombin were obtained from Millipore-Sigma. Antibodies to diphospho–myosin light chain (MLC), pan-MLC, MYPT, pMYPT, p120-catenin, phospho-NFκB, β-actin, and tubulin antibodies were obtained from Cell Signaling (Beverly, MA); Rap1, VE-cadherin, ICAM1, VCAM1, and E-selectin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-linked anti-mouse and anti-rabbit IgG were obtained from Cell Signaling (Beverly, MA). Unless otherwise specified, all biochemical reagents were obtained from Sigma (St. Louis, MO).
3
Immunohistochemical Staining of Adhesion Molecules
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Paraffin sections for immunohistochemical staining were placed on poly-L-lysine-coated slide (Fisher scientific, Pittsburgh, PA, USA). Slides were immunostained by Invitrogen's HISOTO-STAIN-SP kits using the Labeled-(strept) Avidin-Biotin (LAB-SA) method. After antigen retrieval, slides were immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity and rinsed with PBS. After being rinsed, slides were incubated with 10% nonimmune goat serum for 10 min at room temperature and incubated with primary antibodies of ICAM-1, VCAM-1, and E-selectin (1:200; Santa Cruz, CA, USA) in humidified chambers overnight at 4°C. All slides were then incubated with biotinylated secondary antibody for 20 min at room temperature and then incubated with horseradish peroxidase-conjugated streptavidin for 20 min at room temperature. Peroxidase activity was visualized by 3,3′-Diaminobenzidine (DAB; Novex, CA) substrate-chromogen system, counterstaining with hematoxylin (Zymed, CA, USA). For quantitative analysis, the average score of 10~20 randomly selected area was calculated by using NIH Image analysis software, Image J (NIH, Bethesda, MD, USA).
4
Protein Expression Analysis by Western Blot
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Cells were lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 0.1 mg/ml soybean inhibitor). Cell lysates were centrifuged at 15,000 rpm for 10 min at 4℃. Protein concentrations were measured by the BCA method using BSA as the standard. Proteins (30 µg) were separated by 8~10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% fat-free milk for 1 h in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.6, and 150 mM NaCl) containing 0.1% Tween 20 (TBS-T) and then incubated with the following primary rabbit or mouse antibodies: anti-BMP4, -ICAM-1, -VCAM-1 and -E-selectin (all from Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich). The antibodies were diluted 1:500~1:2,000 in 1% fat-free milk in TBS-T and incubated at 4℃ overnight. After washing, the membranes were incubated with secondary peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Bio-Rad Laboratories, Hercules, CA, USA) diluted 1:1,000 in 1% fat-free milk in TBS-T at room temperature for 1 h. Detection was achieved using Pierce ECL Plus Western Blotting Substrate (ThermoFisher Scientific Inc., Rockford, IL, USA).
5
Puerarin Injection for Diabetic Injury
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Puerarin injection was purchased from Hunan WZT Pharmaceutical Co., Ltd. (China). The STZ was obtained from Sigma (USA). The primers for ICAM-1, LOX-1, NADPH oxidase 2 (NOX2), NOX4, and β-actin were purchased from Generay, Inc. (Shanghai, China). The primary antibodies for ICAM-1, LOX-1, and NF-κB p65 were purchased from Proteintech Group, Inc. (Wuhan, China), Abcam (USA), and Boster (Wuhan, China), respectively, whereas those for NOX2, NOX4, and E-selectin were purchased from Santa Cruz Biotechnology.
6
Western Blotting Inflammatory Markers
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Cells and tissue protein lysates were analysed by standard western blotting procedure, using antibodies such as PGE2, iNOS, TNF-α, IL-1β, IL-6, IL-10, NF-κB p65, ICAM-1, VCAM-1, P-selectin, and E-selectin obtained from Santa Cruz Biotechnology (Santa Cruz, CA) [27 (link)].
7
Antibody validation for Western blot and IF
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The following antibodies were used: GFP (Clontech #632381 western blot (WB), 1:3,000), MYC (WB, Abcam # ab9132, 1:5,000), FLAG (WB, Sigma # F1804, 1:500), Lamin A (WB, Sigma-Aldrich # L1293 1:1,000), α-Tubulin (WB, Sigma-Aldrich # T5168, 1:20,000), β-Actin (WB, Cell Signaling Technology # 3700, 1:10,000), GAPDH (WB, Cell Signaling Technology # 5174, 1:5000), NFκB p65 (WB, Santa Cruz Biotechnology # sc-372, 1:1000), VCAM-1 (WB, R&D # BBA19, 1:1000), E-selectin (WB, Santa Cruz Biotechnology # sc-14011, 1:1000), LDLR (WB, BioVision/AH # 3839-100, 1:2,000), Giantin (immunofluorescence (IF), Covance # PRB-114C, 1:500). Fluorophore-conjugated secondary antibodies were from Thermo Fisher Scientific (IRDye® 800CW Donkey anti-Rabbit # SA5-10044, IRDye® 800CW Donkey anti-Mouse SA5- 10170) and LI-COR (IRDye® 800CW Donkey anti-Goat # 926-32214, IRDye® 680RD Goat anti-Rabbit # 926-68071 D). All secondary antibodies were diluted 1:10,000. The following experimental materials were used: FBS (Sigma-Aldrich), penicillin-streptomycin (Termo Fisher Scientific), doxycycline (Clonetech), blasticidin (Termo Fisher Scientific), brefeldin A (Sigma- Aldrich) hygromycin B gold (Sigma-Aldrich), Methyl-beta-cyclodextrin (Sigma-Aldrich), cholesterol (Sigma-Aldrich), IL-1β (R&D # 201-LB- 025), Dual Luciferase® reporter assay system (E1960) was purchased from Promega (Madison, WI, USA).
8
Inflammatory Pathway Modulation Protocol
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TNF-α, antibiotic-antimycotic, fetal bovine serum (FBS), chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein di-acetate (CM-H2DCFDA) and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Invitrogen (Carlsbad, CA, USA). IL-6, VCAM-1, E-selectin, ICAM-1, IκB-α, p-IκB-α, NF-κB, laminB, eNOS, Akt, phospho-eNOS, phosphor-Akt, GTPCH, NLRP3, ASC, caspase-1 and β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from stress gen Biotechnologies Corp & Enzo Life Sciences (Farmingdale, NY, USA). 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from sigma Chemical co. (St. Louis, MO, USA). Human tetrahydrobiopterin ELISA kit was purchased from MyBioSource (San Diego, CA, USA). The other reagents used in this study were the highest purity commercially available.
9
Inflammatory Signaling Pathway Modulation
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Millipore Sigma (Billerica, MA, USA). Recombinant human TNF-α and recombinant human IFN-γ were purchased from Bio-Techne Ltd. (Abingdon, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Primary antibodies against p-IKK α/β (cat no. 2697), NF-κB p65 (cat no. 8242), p-Akt (cat no. 9271), and ICAM-1 (cat no. 4915) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against IKK α/β (cat no. 7607), p-IκB-α (cat no. 8404), IκB-α (cat no. 203), Akt1/2/3 (cat no. sc-8312), PARP (cat no. sc-9542), α-tubulin (cat no. sc-8035), Filaggrin (cat no. sc-66192), VCAM-1 (cat no. sc-1504), E-selectin (cat no. sc-5262), and β-actin (cat no. sc-81178) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories, Inc. (West Grove, PA, USA). The histamine ELISA kit was obtained from Enzo life Sciences, Inc. (Farmingdale, NY, USA). The ELISA kits for TNF-α and IL-6 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).
10
Protein Expression Analysis in HUVEC Cells
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Proteins were extracted from the HUVECs of the different cultures by cell lysis buffer (Cell Signaling Technology, Danvers, MA). Equal amounts of protein were separated by 10% SDS–PAGE gels and then transferred to PVDF membranes (Millipore Corporation). The PVDF membranes were blocked in 5% skim milk at room temperature for 1 h and then incubated with the corresponding primary antibody (1:1000) at room temperature for 2 h. The primary antibodies were monoclonal antibodies against β-actin (ProteinTech, Wuhan, China), PTEN (ProteinTech, Wuhan, China), SET8 (ProteinTech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), forkhead box protein O1 (FOXO1) (Cell Signaling Technology, Danvers, MA), and e-selectin (Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal antibodies against ICAM-1 (Cell Signaling Technology, Danvers, MA) and p-p65 (Cell Signaling Technology, Danvers, MA). The membranes were incubated with the corresponding secondary antibody (1:1000) at room temperature for 1 h. The membranes were then washed, and the proteins were detected by a LAS-4000 mini CCD camera (GE Healthcare).
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