Western Blot Analysis of Inflammasome Proteins

The cells were harvested and lysed using RIPA buffer with protease inhibitor (Thermo Fisher Scientific), and the protein concentrations were determined by the BCA protein assay (Thermo Fisher Scientific). A total of 40 ug of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes as previously described [23 (link)]. Blots were incubated with primary antibodies against NLRP3 (#15101; Cell signaling technology, Danvers, MA, USA), ASC (#67824; Cell signaling technology), pro-IL-1β (sc-32294; 1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), cleaved IL-1β (#83186; Cell signaling technology), pro-CASP1 (#3866; Cell signaling technology), cleaved CASP1 (#89332; Cell signaling technology), and GAPDH (sc-32233; Santa Cruz Biotechnology) at the 1:1,000 dilution, followed by an incubation with the secondary antibody conjugated to HRP for 1 h in room temperature. The bands were visualized using SuperSignal West Pico PLUS Chemiluminescent (Thermo Fisher Scientific) and luminescent image analyzer (ChemiDoc XRS + Systems, Bio-Rad Laboratories) at multiple exposure settings. For quantification, ImageJ software (National Institutes of Health) were used to analyzed the bands intensity.

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Park H.S., Lee B.C., Chae D.H., Yu A., Park J.H., Heo J., Han M.H., Cho K., Lee J.W., Jung J.W., Dunbar C.E., Oh M.K, & Yu K.R. (2024). Cigarette smoke impairs the hematopoietic supportive property of mesenchymal stem cells via the production of reactive oxygen species and NLRP3 activation. Stem Cell Research & Therapy, 15, 145.

Publication 2024

protease inhibitor BCA protein assay NLRP3 pro-IL-1β cleaved IL-1β pro-CASP1 cleaved CASP1 GAPDH SuperSignal West Pico PLUS Chemiluminescent ChemiDoc XRS + Systems

Corresponding Organization :

Other organizations : Seoul National University, Sookmyung Women's University, Korea National Institute of Health, National Heart Lung and Blood Institute, National Institutes of Health

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein levels of NLRP3
Protein levels of ASC
Protein levels of pro-IL-1β
Protein levels of cleaved IL-1β
Protein levels of pro-CASP1
Protein levels of cleaved CASP1

control variables

GAPDH protein levels (used as a loading control)

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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