RIPA lysis buffer (Thermo Fisher) with protease inhibitor (Sigma) was used to extract the total protein. The same amounts of total proteins were separated via SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF, Millipore, Bedford, MA, USA) membranes. After blocking with 5% skim milk, the membranes were incubated with the primary antibodies[TRIM47 (Sigma); Twist, E-cadherin, N-cadherin, ZEB1, SNAI1, SLUG, and vimentin (Abcam); p53, P21, CDK4, CDK6, cyclin D1, p65, P-p65, and GAPDH (CST)] overnight at 4°C and then with the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA). Signals were detected with enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA).
Snai1
Manufactured by Abcam
Sourced in United States
SNAI1 is a gene that encodes the Snail family transcriptional repressor 1 protein. This protein functions as a transcriptional regulator and plays a role in processes such as embryonic development and cell differentiation.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Vimentin, by Abcam (1 mentions)
Enhanced chemiluminescence system, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Twist, by Merck Group (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Gapdh, by US Biological (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Twist1, by Proteintech (1 mentions)
5 protocols using snai1
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting Analysis of Epithelial-Mesenchymal Transition Markers
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Total protein was isolated from cells in RIPA lysis buffer on ice. Protein concentration was quantified using a Brad-ford Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were boiled before being separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Pall Corp, Port Washington, NY, USA). The following primary antibodies were used in the immunoblotting assays: SNAI1 (ab117866; Abcam), E-cadherin (24E10, number 3195; Cell Signaling, Beverly, MA, USA), vimentin (C-20, sc-7557; Santa Cruz Biotechnology Inc, Dallas, USA, USA), and GAPDH (G8140; US Biological, Swampscott, MA, USA). Horseradish peroxidase–conjugated secondary antibodies (Bio-Rad Laboratories) were used at a 1:1000–1:5000 dilution and detected using Western Blotting Luminol Reagent (sc-2048; Santa Cruz Biotechnology Inc), as described in our previous study.20 (link)
3
FBXO11 Mutant Lung Development Analysis
4
Western Blot Analysis of Signaling Proteins
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Total proteins were extracted from cells with RIPA buffer (Sigma Aldrich), separated by SDS‐PAGE gels and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were blocked with 5% nonfat milk and incubated with primary antibodies (β‐CATENIN, AKT, pAKT, mTOR, pmTOR, MEK, pMEK (Cell Signaling Technology), MMP2, SNAI1 (Abcam, Cambridge, UK), IL32, TWIST1, GAPDH (Proteintech) overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. The immunoreactive protein bands were detected by ECL detection system. GAPDH was used as a loading control.
5
Western Blot Analysis of EMT Markers
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Treated cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (KenGen, China), and protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime, China). Total protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in 5% non-fat milk and incubated with primary antibodies against SP1 (1:1000; Bioworld Technology, USA), SNAI1, CDH1, CDH2, VIM and matrix metalloproteinase-2 (MMP-2; 1:1000 each; all from Abcam, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology, USA). GAPDH served as a loading control (1:5000; Bioworld Technology, USA).
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