Western blotting analyses were performed following standard protocols. Briefly, cells were lysed in RIPA Lysis Buffer (Beyotime, Jiangsu, China), which contained Protease Inhibitor Cocktail (Roche, Mannheim, Germany). Protein concentrations were measured using a BCA Protein Assay Reagent (Thermo, MA, USA). Equal amounts of cell lysate were loaded onto SDS-PAGE gels and then transferred to PVDF membranes. Membranes were blocked with 5% fat-free milk and incubated with primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated species-specific secondary antibodies. Bands were visualized with enhanced chemiluminescence reagent (Millipore, MA, USA). The following commercial antibodies were used in this study: Nr5a2 (1:1000, Sigma, MO, USA), E-cadherin, N-cadherin, Twist1, Vimentin, MMP-2, beta-Catenin, Wnt3a, c-Myc and Cyclin D1 (1:1000, Cell Signaling Technology, MA, USA), and Snail2 and GAPDH (1:1000, Proteintech, Wuhan, China).
Snail2
Manufactured by Proteintech
Sourced in United States
Snail2 is a laboratory equipment product offered by Proteintech. It is designed for specific research applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information about the intended use or features of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Proteintech (4 mentions)
Gapdh, by Proteintech (3 mentions)
Snail1, by Proteintech (3 mentions)
N cadherin, by Proteintech (2 mentions)
Fibronectin, by Proteintech (2 mentions)
E cadherin, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Twist1, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Wnt3a, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
5 protocols using snail2
1
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
2
Immunohistochemical Analysis of EMT Markers
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IHC was performed as described previously [19 (link), 20 (link)]. Briefly, tissue sections were deparaffinized and dehydrated, incubated with H2O2, subjected to high-pressure repair, and blocked with BSA. anti-Numb (Abcam, Cambridge, UK, dilution: 1:400), Snail1 (Proteintech, Chicago, IL, dilution: 1:200), Snail2 (Proteintech, dilution: 1:200), E-cad (Abcam, 1:1000), N-cad (Proteintech, dilution: 1:200), Vimentin (Proteintech, dilution: 1:500), and Ki67 (Proteintech, dilution: 1:500) overnight. Slices were covered with the secondary antibody, detected with DAB, co-stained with hematoxylin, and evaluated by two professional pathologists for the staining scores.
3
Western Blot Analysis of EMT Markers
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Western blotting analysis was conducted as described previously in our study (16 (link)). Total cellular proteins were harvested in IP lysis buffer (Beyotime, Shanghai, China) with a cocktail of proteinase and phosphatase inhibitors (Bimake.cn) and were measured by a BCA protein assay kit (Thermo Fisher Scientific, USA). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membrane (Millipore, USA). Then the membranes were blocked with 5% skimmed milk in Tris-Buffered saline (TBS) for 1 hour at RT and incubated with primary antibodies overnight at 4°C followed by incubation with DyLight fluorescent dye labelled secondary antibodies for 1 hour at RT. Finally, immunoblot signals were detected using an Odyssey Imaging System (LI-COR Biosciences, USA). Primary antibodies used in this analysis were listed as follows: TRIM50 (1:100 dilution, Abcam, ab174880), E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), Snail1 (1:1000, Proteintech, 13099-1-AP), Snail2 (1:2000, Proteintech, 12129-1-AP), Twist1 (1:1000, Proteintech, 25465-1-AP), Twist2 (1:1000, Proteintech, 11752-1-AP), ZEB1 (1:1000, Proteintech, 21544-1-AP), ZEB2 (1:2000, Proteintech, 14026-1-AP), and β-actin (1:5000, MultiSciences, ab008).
4
Western Blotting of Pancreatic Cancer Cells
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Pancreatic cancer cells were seeded in 12-well plates (1 × 105 cells/well) and harvested 48 h after transfection. Proteins were extracted from tissues or cells using RIPA Lysis buffer supplemented with phenylmethylsulfonyl fluoride, protease inhibitor, phosphatase inhibitor. Protein concentration was quantified using bicinchoninic acid (BCA) protein concentration determination kit (TaKaRa, Japan). Samples were then resolved on 10% SDS–polyacrylamide gels and transferred to nitrocellulose. PVDF membranes were blocked with 5% skimmed milk for 2 h, then incubated overnight with primary antibodies against: E-cadherin (Abcam, Cambridge, UK), N-cadherin (Proteintech, Rosemont, IL, USA), Fibronectin (Proteintech, Rosemont, IL, USA), Vimentin (Proteintech, Rosemont, IL, USA), Snail2 (Proteintech, Rosemont, IL, USA), ZEB1 (Proteintech, Rosemont, IL, USA), Numb (Abcam, Cambridge, UK), ATP11A (Abcam, Cambridge, UK), SRPK2 (Abcam, Cambridge, UK) and GAPDH (Proteintech, Rosemont, IL, USA). The second day, secondary antibodies incubations were performed using horseradish peroxidase(HRP)-conjugated secondary antibodies (Proteintech, Rosemont, IL, USA). Finally, an ECL-chemiluminescent kit (Thermol Biotech Inc, Waltham, MA, USA) was used to detect the protein bands.
5
TGF-β1-Smad2/3-Snail Signaling Pathway
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Whole-cell lysates were prepared from PC specimens and cell lines. Cells were pretreated with 10 ng/ml of TGF-β1 (Peprotech, RockyHill, New Jersey, USA) plus Notch signaling inhibitor RO4929097 (10 μM, Selleckchem, USA) for 24 h to detect the activity of TGF-β1-Smad2/3-Snail signaling. All samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes (Millipore Corp, Bedford, MA, USA), and incubated with primary Numb (Abcam,1:1000), ZEB-1 (Proteintech, 1:1000), Fibronectin (Proteintech, 1:1000), E-cadherin (E-cad, Abcam, dilution: 1:1000), N-cadherin (N-cad, Proteintech, 1:1000), Vimentin (Proteintech, dilution: 1:1000), Smad2/3 (Cell Signaling Technology, Beverly, USA, dilution: 1:500), p-ERK (Cell Signaling Technology, dilution: 1:1000), Snail1 (Proteintech, 1:500), Snail2 (Proteintech, 1:500), cleaved-Notch1 (Cell Signaling Technology, dilution: 1:1000), phosphorylating EGFR at tyrosine 1045 (p-EGFR1045, Cell Signaling Technology, dilution: 1:1000) and GAPDH (Proteintech, 1:3000) overnight at 4 °C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Proteintech) for 2 h at room temperature. Immunoreactive protein bands were visualized with an ECL detection kit (Thermol Biotech Inc, USA). Each experiment was repeated three times.
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