As described by Xie et al [14 (link)], all proteins were isolated by the RIPA buffer (Solarbio, Beijing, China) and the concentration of protein was tested using a BCA Protein Assay Kit (Solarbio). The proteins were subjected to 10% SDS-PAGE, followed by transfer of the separated proteins onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The main antibodies were ubiquitin-specific peptidase 2 (Proteintech, Rosemont, IL, USA, 1:1000), GAPDH (Proteintech, 1:5000), nuclear factor (NF)-κB p65 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), phospho-NF-κB p65 (Cell Signaling Technology, 1:1000), N-cadherin (Proteintech, 1:5000), E-cadherin (Proteintech, 1:25,000), Vimentin (Proteintech, 1:5000), and Snail (Cell Signaling Technology, 1:1000). Secondary antibodies used were horseradish peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) and horseradish peroxidase-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, 1:8000). Incubations with the antibodies were performed per the manufacturer’s instructions.
Protocol detail
Protein Isolation and Western Blot Analysis
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RIPA buffer
BCA Protein Assay Kit
polyvinylidene fluoride membranes
GAPDH
nuclear factor (NF)-κB p65
phospho-NF-κB p65
N-cadherin
E-cadherin
Vimentin
Snail
Anti igg
Antibodies
Assay
Buffer
E cadherin
Gapdh
Goat
Horseradish peroxidase
Membranes
Mouse
N cadherin
Nuclear factor κb
Polyvinylidene fluoride
Proteins
Rabbit
Ripa
Sds page
Snail
Ubiquitin specific peptidase
Vimentin
Corresponding Organization : First Affiliated Hospital of Zhengzhou University
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Variable analysis
independent variables
- None explicitly mentioned
- Protein levels of ubiquitin-specific peptidase 2
- Protein levels of GAPDH
- Protein levels of NF-κB p65
- Phosphorylation levels of NF-κB p65
- Protein levels of N-cadherin
- Protein levels of E-cadherin
- Protein levels of Vimentin
- Protein levels of Snail
- Protein isolation using RIPA buffer
- Protein concentration measurement using BCA Protein Assay Kit
- Protein separation by 10% SDS-PAGE
- Protein transfer to polyvinylidene fluoride membranes
- Primary antibody concentrations for ubiquitin-specific peptidase 2 (1:1000), GAPDH (1:5000), NF-κB p65 (1:1000), phospho-NF-κB p65 (1:1000), N-cadherin (1:5000), E-cadherin (1:25,000), Vimentin (1:5000), and Snail (1:1000)
- Secondary antibody concentrations for horseradish peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) and horseradish peroxidase-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (1:8000)
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