The experimental steps of Western blot were conducted by our previous literature.21 (link) The primary antibodies listed below were incubated with the membranes at 4 °C and stayed overnight (at a 1:1,000 dilution): α-SMA, transforming growth factor (TGF)-β1, collagen-I, collagen-III, MMP-2, MMP-9, NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-17A, and IL-18 (ABclonal Technology, Wuhan, China); ODC, SSAT, p-Smad-2, t-Smad-2, p-Smad-3, t-Smad-3, and Smad-7 (Cell Signaling Technology, Danvers, Massachusetts); and ubiquitin, β-actin, and β-tubulin (Santa Cruz Biotechnology). The membranes were incubated for 1 h at room temperature with secondary antibodies (diluted at 1:10,000, Proteintech, Wuhan, China). The specific complexes treated with an ECL kit (MultiSciences, Hangzhou, China) were detected using a multiplex fluorescent imaging system.
Protocol detail
Western Blot Analysis of Key Signaling Pathways
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α-SMA
collagen-I
NLRP3
Caspase-1
GSDMD-N
IL-17A
IL-18
p-Smad-2
t-Smad-2
p-Smad-3
t-Smad-3
Smad-7
ubiquitin
β-actin
β-tubulin
ECL kit
Actin
Antibodies
Caspase 1
Collagen
Collagen i
Dilution
Interleukin 17a
Interleukin 18
Interleukin 1β
Membranes
Mmp 2
Mmp 9
Smad 3
Smad 7
Ssat
Transforming growth factor β1
Tubulin
Ubiquitin
Western blot
Corresponding Organization :
Other organizations : Harbin Medical University, Mudanjiang Medical University, COMSATS University Islamabad
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Variable analysis
independent variables
- Primary antibodies: α-SMA, transforming growth factor (TGF)-β1, collagen-I, collagen-III, MMP-2, MMP-9, NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-17A, IL-18 (ABclonal Technology, Wuhan, China)
- ODC, SSAT, p-Smad-2, t-Smad-2, p-Smad-3, t-Smad-3, and Smad-7 (Cell Signaling Technology, Danvers, Massachusetts)
- Ubiquitin, β-actin, and β-tubulin (Santa Cruz Biotechnology)
- Not explicitly mentioned
- Primary antibodies incubated with the membranes at 4 °C and stayed overnight (at a 1:1,000 dilution)
- Membranes incubated for 1 h at room temperature with secondary antibodies (diluted at 1:10,000, Proteintech, Wuhan, China)
- Specific complexes treated with an ECL kit (MultiSciences, Hangzhou, China) and detected using a multiplex fluorescent imaging system
- No positive or negative controls explicitly mentioned
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