Whole cell lysates were made in Aqueous Lysis Buffer (50mM Tris pH 7.5, 150mM NaCl, 10mM EDTA pH8.0, 0.2% Sodium azide, 50mM NaF, 0.5%NP40) and resolved on a gradient gel as previously described(20 (link)). Primary antibodies used were E-cadherin, Vimentin, Zeb1, Snail, Slug, Sox9, GAPDH, CSF1, Galectin-3, MASP1/3, Fibronectin (BD Biosciences)
Snail
Manufactured by BD
Sourced in United States
The Snail is a compact and versatile lab equipment designed for a variety of applications. It features a durable construction and precise temperature control capabilities. The core function of the Snail is to provide a controlled environment for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by BD (4 mentions)
N cadherin, by BD (3 mentions)
E cadherin, by BD (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Gapdh, by BD (1 mentions)
Galectin 3, by BD (1 mentions)
Fibronectin, by BD (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Promega (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti vimentin, by BD (1 mentions)
Claudin 7, by Santa Cruz Biotechnology (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Phospho erk1 2, by BD (1 mentions)
8 protocols using snail
1
Whole Cell Lysate Western Blot
2
Protein Expression Analysis by Western Blot
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Total protein was extracted from cultured cells and analyzed as previously described 27 (link), 28 (link). A total of 50 μg of protein extracts were loaded to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). After incubated with the primary antibodies overnight at 4 ℃ and then with HRP conjugated secondary antibodies (Promega, USA) for 1 h at room temperature, the membranes were subjected to the ECL chemiluminescence system (Pierce, USA). All of the experiments were repeated thrice. Primary antibodies used: CBX8 from Sigma Aldrich; Snail, Slug and Vimentin from BD Biosciences; E-cadherin and N-cadherin from Cell Signaling Technology; CBX6, Fibronectin and GAPDH from Santa Cruz.
3
Antibody Sources for Western Blotting
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Human anti-TEL2 antibody was obtained from Sigma (HPA029033). Human anti-SERPINE1 antibody was obtained from Santa (sc-5297). Other primary antibodies used for western blotting, such as anti-E-cadherin, N-cadherin, Snail and anti-Vimentin, were obtained from BD Company. Anti-HA was obtained from Cell Signaling Technology.
4
Immunofluorescence Visualization of Cell Junctions
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Immunofluorescence was performed as previously described (24 (link)). The cells were fixed with cold methanol for 10 min and blocked with 1% bovine serum albumin (BSA; Rocky Mountain Biologicals, Inc.) solution. Briefly, antibodies of E-cadherin (cat. no. 610182; 1:500; BD Biosciences), Snail (cat. no. sc-271977; 1:500; Santa Cruz Biotechnology, Inc.) and claudin-7 (cat. no. ab207300; 1:500; Abcam) were used. The cells were reacted with Cy2-conjugated goat anti-mouse IgG (cat. no. 111-223-003; 1:500; Jackson ImmunoResearch Laboratories, Inc.) and Cy3-conjugated goat anti-rabbit IgG (cat. no. 111-156-003; 1:500; Jackson ImmunoResearch Laboratories, Inc.). The cell nuclei were stained with 4′,6′-diamidino-2-phenylindole dihydrochloride (cat. no. v62249; 1:1,000; Thermo Fisher Scientific, Inc.) at RT for 5 min. Fluorescence images were captured using confocal microscopy (LSM710 Carl Zeiss AG).
5
Western Blot Analysis of Signaling Proteins
6
Antibody Panel for EMT Evaluation
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The antibodies used in this paper are detailed below:
N-cadherin (CST, cat# 13116), E-cadherin (CST, cat# 14472), PCNA (CST, cat# 13110), Slug (CST, cat# 9585), Snail (CST, cat# 3879), vimentin (CST, cat# 5741), β-actin (CST, cat# 3700), KRAS (CST, cat# 67648), p-AKT (473) (CST, cat# 4060), p-AKT (CST, cat# 9275), IGF2BP2 (CST, cat# 14672), HA (CST, cat# 5017), CD44 (BD Pharmingen; IM7) and C-myc (CST, cat# 18583).
N-cadherin (CST, cat# 13116), E-cadherin (CST, cat# 14472), PCNA (CST, cat# 13110), Slug (CST, cat# 9585), Snail (CST, cat# 3879), vimentin (CST, cat# 5741), β-actin (CST, cat# 3700), KRAS (CST, cat# 67648), p-AKT (473) (CST, cat# 4060), p-AKT (CST, cat# 9275), IGF2BP2 (CST, cat# 14672), HA (CST, cat# 5017), CD44 (BD Pharmingen; IM7) and C-myc (CST, cat# 18583).
7
Comprehensive Protein Expression Analysis
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Total protein was obtained by a RIPA buffer (Beyotime, Shanghai, P.R. China) and quantified with bicinchoninic acid protein assay kit (Solarbio, Shanghai, P.R. China).6 (link) The same quantity of protein (30 µg per lane) was separated by 10% SDS-PAGE. The proteins were incubated with primary antibodies overnight at 4°C against C14orf159 and GAPDH (1:200 and 1:3,000; Sigma-Aldrich Co.), p-ERK, ERK, p-P90RSK, P90RSK, p-P38, P38, p-P65, P65, p-AKT, AKT, Cyclin A2, Cyclin B1, Cyclin D1, Myc-tag, Vimentin, Snail, Slug (1:1,000; BD Transduction Laboratories, Lexington, KY, USA), E-cadherin, N-cadherin, Zo-1 and Occludin (1:1,000; Cell Signaling Technology, Danvers, MA, USA). After incubating with anti-mouse or anti-rabbit IgG (BD Transduction Laboratories) at 37°C for 2 hours, the membranes were developed with enhanced chemiluminescence reagent (Solarbio).
8
Immunoblotting of Epithelial-Mesenchymal Transition Markers
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Verteporfin (cat. no. SML0534) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Both BAY 11-7082 (cat. no. 10010266) and TNF-α (cat. no. 32020) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Protease inhibitor cocktail was purchased from Thermo Fisher Scientific (cat. no. 78430, Thermo Fisher Scientific Inc., Waltham, MA, USA). The primary antibodies for CARMA3 (cat. no. ab137383; Abcam, Cambridge, UK); E-cadherin (cat. no. sc-7870; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); N-cadherin (cat. no. 14215; Cell Signaling Technology, Danvers, MA, USA); Fibronectin (cat. no. 610077; BD Transduction Laboratories); Snail (cat. no. 3879; Cell Signaling Technology, Danvers, MA, USA); Slug (cat. no. 9585; Cell Signaling Technology, Danvers, MA, USA); YAP (cat. no. 14074; Cell Signaling Technology, Danvers, MA, USA) and α-Tubulin (cat. no. T-5168; Sigma-Aldrich, St. Louis, MO, USA) were used. All secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
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