Protein was extracted from liver tissue or cell cultures as described (22 (link)). Protein was extracted from liver tissue or cell cultures and subjected to 4-20% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membrane (Bio-Rad). The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). The SIRPα, p-GSK3β, GSK3β, NICD, β-catenin, XBP1 , ASC, cleaved caspase-1, Lamin B2, β-actin (Cell Signaling Technology), CD47, SMO, Dvl2, NRX (Santa Cruz Biotechnology), Gli1 (Invitrogen), NEK7, and NLRP3 (Abcam) mAbs were used. The membranes were incubated with Abs, and then added Western ECL substrate mixture (Bio-Rad) for imaging with the iBright FL1000 (ThermoFisher Scientific). Relative quantities of protein were determined by comparing the β-actin expression using iBright image analysis software (ThermoFisher Scientific). See Supplementary Materials .
Protocol detail
Western Blot Analysis of Protein Extraction
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nitrocellulose membrane
NE-PER Nuclear and Cytoplasmic Extraction Reagents
SIRPα
p-GSK3β
GSK3β
β-catenin
cleaved caspase-1
Lamin B2
β-actin
NLRP3
Western ECL substrate mixture
iBright FL1000
iBright image analysis software
Actin
Caspase 1
Cd47
Cell cultures
Cytoplasmic
Cytosolic
Gsk3β
Lamin b2
Mabs
Membranes
Nitrocellulose
Protein
Sds polyacrylamide gel electrophoresis
Sirpα
Xbp1
β catenin
Corresponding Organization : University of California, Los Angeles
Other organizations : Tianjin Medical University, Nankai University, Tianjin First Center Hospital, Tianjin Medical University General Hospital, Renji Hospital
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Variable analysis
independent variables
- Protein extraction from liver tissue or cell cultures
- Expression levels of SIRPα, p-GSK3β, GSK3β, NICD, β-catenin, XBP1, ASC, cleaved caspase-1, Lamin B2, β-actin, CD47, SMO, Dvl2, NRX, Gli1, NEK7, and NLRP3
- Preparation of nuclear and cytosolic fractions using NE-PER Nuclear and Cytoplasmic Extraction Reagents
- Use of specific monoclonal antibodies (mAbs) for protein detection
- Normalization of protein quantities to β-actin expression
- Positive control: β-actin (Cell Signaling Technology)
- Negative control: Not explicitly mentioned
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