Western blot was performed as previously described [25 (link)]. Briefly, lung tissue sample was homogenized using Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total tissue protein purified from lungs were separated by SDS-PAGE gels and then transferred to 0.22-μm PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies against NF-kB p65 (1:500 dilution, Abcam), IκBα (1:1000 dilution, CST), TGF-β (1:1000 dilution, Abcam), pSmad2 (1:1000 dilution, Abcam), α-Tubulin (1:1000 dilution, CST), or GAPDH (1:1000 dilution, Abcam), respectively; and followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako). The expression was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific). Semiquantitative analysis was done using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences).
Protocol detail
Quantitative Western Blot Analysis of Lung Tissue
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Multi-beads shocker
T-PER reagent
proteinase and dephosphorylation inhibitors
0.22-μm PVDF membranes
NF-kB p65
TGF-β
pSmad2
GAPDH
horseradish peroxidase-conjugated secondary antibodies
enhanced chemiluminescence detection kit
ImageQuant LAS 4000 mini
Antibodies
Chemiluminescence
Dilution
Gapdh
Gels
Horseradish peroxidase
Inhibitors
Iκbα
Lungs
Membranes
Nf kb
Protein
Proteinase
Pvdf
Sds page
Tgf β 1
Tissue
Western blot
α tubulin
Corresponding Organization : Nagasaki University
Other organizations : Tohoku University
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Variable analysis
independent variables
- None explicitly mentioned
- NF-kB p65 expression
- IκBα expression
- TGF-β expression
- PSmad2 expression
- α-Tubulin expression
- GAPDH expression
- Tissue sample preparation (homogenization and protein extraction)
- SDS-PAGE separation and PVDF membrane transfer
- Antibody dilutions and incubation conditions
- Chemiluminescence detection method
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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