Western Blotting Assay Protocol

Western blotting assays were carried out as described previously (Du et al., 2017 (link)). Primary antibodies in the experiments were applied as follows: ICAM-1 (diluted at 1:1,000, ProteinTech, Chicago, IL, United States), VCAM-1 (diluted at 1:1,000, ProteinTech), E-selectin (diluted at 1:1,000, R&D Systems, Minneapolis, MN, United States), β-tubulin (diluted at 1:1,000, ProteinTech), NF-κB-p65 (diluted at 1:1,000, CST, Danvers, MA, United States), NF-κB-pp65 (diluted at 1:1,000, CST), IκBα (diluted at 1:1,000, CST), and phospho-IκBα (diluted at 1:1,000, CST). Then the secondary antibodies with horseradish peroxidase coupling (1:10,000 dilution) obtained from ProteinTech were used for 2 h at room temperature. Electrochemiluminescence (ECL) detection reagents were purchased from Millipore (Billerica, MA, United States). Target proteins in the membrane were visualized with a Bio-Rad (Hercules, CA, United States) exposure imaging system.

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Hu J., Chen R., An J., Wang Y., Liang M, & Huang K. (2021). Dauricine Attenuates Vascular Endothelial Inflammation Through Inhibiting NF-κB Pathway. Frontiers in Pharmacology, 12, 758962.

Publication 2021

ICAM-1 VCAM-1 E-selectin β-tubulin ECL) detection reagents

Antibodies Dilution E selectin Horseradish peroxidase Icam 1 Membrane proteins Nf κb Nf κb p65 Phospho Pp65 Tubulin Vcam 1 Western blotting assays

Corresponding Organization : Union Hospital

Other organizations : Handan College, Sun Yat-sen University, The First Affiliated Hospital, Sun Yat-sen University

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Variable analysis

independent variables

Primary antibodies: ICAM-1, VCAM-1, E-selectin, NF-κB-p65, NF-κB-pp65, IκBα, phospho-IκBα

dependent variables

Protein expression levels of ICAM-1, VCAM-1, E-selectin, NF-κB-p65, NF-κB-pp65, IκBα, phospho-IκBα

control variables

β-tubulin (diluted at 1:1,000, ProteinTech)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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