Protein Expression Analysis Using Western Blot

The cells were lysed and homogenized using radioimmunoprecipitation assay buffer (Cell Signaling Technology), and the protein concentration was measured by an Enhanced Bicinchoninic acid Protein Assay Kit (Beyotime). Equal amounts of protein extracts were separated by electrophoresis on a 12% NuPAGE Bis-Tris Mini Gel (Invitrogen) and then transferred to nitrocellulose membranes (GE Healthcare) (24 (link)). The membranes were blocked for 1 hour in Tris-buffered saline containing 5 % bovine serum albumin and then probed with antibodies. Antibodies against SRM (Cat#: 19858), ODC1 (Cat#: 17003), SMOX (Cat#: 15052), iNOS (Cat#: 18985), IL-6 (Cat#: 21865), and GAPDH (Cat#: 60004) were purchased from Proteintech, and nuclear factor kappa B (NF-κB) p65 (Cat#: 8242S) was purchased from Cell Signaling Technology. The membranes were then probed with secondary antibodies conjugated to horseradish peroxidase. Next, the immunoblots were developed as recommended in the ECL Advance Western blot detection kit (Amersham Biosciences).

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Li C., Zhang W., Chang X., Di X., Xie Q., Lin B., Zhang H., Ye Z., Lan M., Lian J., Zhang H., Qiu X., Zeng J, & Huang M. (2023). The upregulation of peripheral blood polyamine metabolites spermidine and spermine in children with hand, foot, mouth disease is related to enterovirus 71 capsid protein VP1, but not VP4. Translational Pediatrics, 12(2), 194-207.

Publication 2023

radioimmunoprecipitation assay buffer Enhanced Bicinchoninic acid Protein Assay Kit NuPAGE Bis-Tris Mini Gel nitrocellulose membranes IL-6 GAPDH nuclear factor kappa B (NF-κB) p65 ECL Advance Western blot detection kit

Antibodies Assay Bicinchoninic acid Bis tris Bovine serum albumin Buffer Cells Electrophoresis Gapdh Horseradish peroxidase Immunoblots Inos Membranes Nf κb p65 Nitrocellulose Nuclear factor kappa b Protein Radioimmunoprecipitation assay Saline Western blot

Corresponding Organization : Guangdong Medical College

Other organizations : Baoding People's Hospital, Guangzhou Medical University, Guangzhou First People's Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of SRM, ODC1, SMOX, iNOS, IL-6, and NF-κB p65

control variables

GAPDH expression as a loading control

controls

Positive control: None mentioned
Negative control: None mentioned

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