pLKO.1 lentiviral vectors harboring short hairpin RNAs (shRNAs) targeting Src or green fluorescent protein (GFP) were obtained from Open Biosystems (Huntsville, AL). Saracatinib and dasatinib were purchased from Selleckchem (Houston, TX). For Western blot, antibodies that recognize p-Src (Tyr416) and Src were purchased from Cell Signaling Technology (Beverly, MA). β-Actin antibody was purchased from Sigma-Aldrich (St Louis, MO). Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (#9782) and Tight Junction Antibody Sampler Kit (#8683) were purchased from Cell Signaling Technology (Beverly, MA).
Epithelial mesenchymal transition antibody sampler kit
Manufactured by Cell Signaling Technology
Sourced in United States
The Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit provides a set of antibodies to detect proteins involved in the EMT process. The kit includes primary antibodies against E-cadherin, N-cadherin, Snail, Slug, Twist, and Vimentin. These antibodies can be used to analyze the expression and localization of EMT-related proteins in cellular and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Saracatinib, by Selleck Chemicals (1 mentions)
Dasatinib, by Selleck Chemicals (1 mentions)
Shrna, by Thermo Fisher Scientific (1 mentions)
Tight junction antibody sampler kit, by Cell Signaling Technology (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Phosphor erk1 2, by Cell Signaling Technology (1 mentions)
Igf 1 receptor β, by Cell Signaling Technology (1 mentions)
Phospho jak2 tyr1007 1008, by Cell Signaling Technology (1 mentions)
Odyssey sa infrared imager, by LI COR (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Anti rabbit, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by Santa Cruz Biotechnology (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
19 protocols using epithelial mesenchymal transition antibody sampler kit
1
Lentiviral Knockdown and Western Blot Analysis
2
Western Blot Analysis of Signaling Proteins
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For western blot analysis, differentially treated cells were washed with cold PBS and then lysed in lysis buffer containing protease and phosphatase inhibitor. Insoluble cell fragments were removed by centrifugation. Cells were separated by SDS-PAGE electrophoresis and next transferred to PVDF membranes. Membranes were incubated overnight with the primary antibodies against phospho-AKT (Cell Signaling Technologies), ERK-1/2 (Cell Signaling Technologies), phosphor-ERK-1/2 (Cell Signaling Technologies), STAT3 (Cell Signaling Technologies), phospho-STAT3 (Cell Signaling Technologies), phospho-IGF-I (Cell Signaling Technologies, Danvers, MA, USA), JAK1, phospho-Jak1 (Tyr1022/1023) (Cell Signaling Technologies), JAK2 (Cell Signaling Technologies), phospho-Jak2 (Tyr1007/1008) (Cell Signaling Technologies), IGF-I Receptor β (Cell Signaling Technologies), AKT (Cell Signaling Technologies), epithelial-mesenchymal transition (EMT) antibody sampler kit (Cell Signaling Technologies). Fluorescent secondary anti-mouse (Thermo Scientific, Waltham, USA) and anti-rabbit (Thermo Scientific, Waltham, USA) antibodies were used and were detected using Odyssey Sa infrared imager (LI-COR Biosciences, Lincoln, USA).
3
Western Blot Analysis of EMT and AMPK Pathway
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Cells were homogenized in 0.2 ml ice cold lysis buffer (50 mM Tris-HCl, pH 7.4, 4% SDS, 20% Glycerol, 2% Mercaptoethanol). After sufficient lysis, the lysate was centrifuged at 12,000 rpm for 5 min at 4°C and the supernatant was collected. The protein concentration was determined using BCA assay. Proteins after lysis were separated in 10% SDS-PAGE gel and transferred onto PVDF membranes for immunoblotting analysis. Then, the membranes were soaked in 40-50ml TBST solution with 5% nonfat milk for 1 h at room temperature, and then incubated with primary antibodies: PDLIM5 (#:10530-1-AP, Proteintech); Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (#:9782, Cell Signaling)); AMPKα (#:5831, Cell Signaling); Phospho-AMPKα(Thr172) (#:2535, Cell Signaling);GAPDH (#:10494-1-AP, Proteintech) at 4°C overnight. After three washes with 40-50ml TBST solution, the transferred membranes were incubated with 1:5000 horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. The protein signals were detected in ECL plus TM Western blotting system kit. GAPDH protein was used as internal reference standard.
4
Western Blot Analysis of EMT Markers
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Cells were lysed in western and IP cell lysis buffer (GenStar BioSolutions) that was supplemented with phenylmethylsulfonyl fluoride (Roche Diagnostics) for 10 min at 4 °C, and then centrifuged at 12,000 ×g for 10 min at 4 °C. The total protein of the supernatants was measured with the Bicinchoninic Acid Protein Assay Kit (GenStar BioSolutions). The protein extracts were electrophoresed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (GE Healthcare Life Sciences). After blocking, the membranes were incubated at 4 °C overnight with the following primary antibodies: Mouse anti-TPM3 (1:1,000; Abcam), rabbit anti-MMP2 (1:1,000; Abcam), rabbit anti-MMP9 (1:1,000; Abcam), mouse anti-tubulin (1:5,000; Abcam), mouse anti-β-actin (1:1,000; Abcam), and an Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (Cell Signaling Technology, Inc.). After 3 washes of 10 min each in tris-buffered saline and polysorbate (TBST; cat. No. 54124; Thermo Fisher Scientific, Inc.), the membranes were incubated with secondary antibodies (cat. No. 78543; Abcam, Inc.) at 1:2,000 dilution for 1 h at 25 °C, and an enhanced chemiluminescence reagent (GenStar BioSolutions) was used to develop the blots.
5
Western Blot Analysis of Wnt/β-Catenin Pathway
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Cells were washed twice with cold phosphate buffered saline (PBS) and lysed in Laemmli sample buffer (Bio‐Rad) with a protease inhibitor, completely EDTA free (Roche). Protein concentration was estimated, using a BCA Protein Assay kit (Pierce, Rockford, MA, USA). Here, 30 μg proteins were electrophoresed on 6% denaturing SDS polyacrylamide gel, and transferred to polyvinylidene fluoride membranes (ImmobilonP; Millipore, Bedford, MA, USA). The membranes were blocked with nonfat milk and 0.1% Tween 20 in Tris‐buffered saline, then, incubated with the primary antibodies overnight at 4°C. The primary antibodies were as the following: SMG‐1(SIGMA, USA), β‐actin(Cell Signaling Technology, Inc, USA), Wnt/β‐Catenin Activated Targets Antibody Sampler Kit(Cell Signaling Technology, Inc, USA) and Epithelial‐Mesenchymal Transition (EMT) Antibody Sampler Kit(Cell Signaling Technology, Inc, USA). Secondary antibodies were incubated at room temperature for 1 hour. Signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
6
Western Blot Analysis of Protein Extraction
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Cells were harvested to extract proteins, followed by Western blot as described previously.14 (link) Briefly, proteins (30 μg each) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels, electrophoresed, and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked for 1 h in 5% skim milk followed by incubation with primary antibodies at 4 °C overnight. The primary antibodies used were as follows: S100A16 (1:500 dilution, ab130419, Abcam, US), epithelial-mesenchymal transition (EMT) antibody sampler kit (1:1,000 dilution, #9782, Cell Signaling Technology, US), Phospho-AKT pathway antibody sampler kit (1:1,000 dilution, 9916T, Cell Signaling Technology, US), Bcl-2 (1:500 dilution, #2870S, Cell Signaling Technology, US), Bax (1:1,000 dilution, #2772S, Cell Signaling Technology, US) and GAPDH (1:2,500 dilution, MBL, Japan). After three washes with TBST, the membranes were incubated with secondary antibodies conjugated to LI-COR IRDye for 1 h at room temperature and were then detected using an Odyssey Imager (LI-COR Biosciences, Lincoln, NE).
7
Antibody-Based EMT Pathway Modulation
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Lipofecatmine 2000 for cell transfection was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against MAZ, ZEB1 and ZEB2 were purchased from Abcam (Cambridge, MA, USA). Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit was from Cell Signaling technology (Danvers, MA, USA). Anti-β-actin antibody was from Signalway Antibody LLC (College Park, Maryland, USA). Unless otherwise noted, all other reagents were from Sigma-Aldrich (St. Louis, MO, USA).
8
Epithelial-Mesenchymal Transition Western Blot Protocol
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Cells were washed with ice-cold PBS, and lysed using Qproteome Mammalian Protein Prep Kit (Qiagen, Hilden, Germany) or by lysis buffer containing 20 mM Tris/HCl pH 8.0, 150 mM KCl, 1 mM EDTA, 0.2 mM Na3VO4, 1% Triton X-100, 0.5 mM PMSF with protein inhibitor cocktail (complete, Roche Applied Science, Basel, Switzerland). Equal amounts of protein samples were denatured at 95°C for 5 minutes, separated by 10% SDS-PAGE and transferred onto PDVF membranes. Membranes were then blocked with 5% non-fat dry milk in PBS/0.1% Tween-20 and incubated overnight with the following antibodies: ZO-1 mAB, Vimentin XP rabbit mAb, E-cadherin rabbit mAb, Snail rabbit mAb, Slug rabbit mAb, TCF8/ZEB1 Rabbit mAb, β-Catenin rabbit mAb Anti-rabbit IgG, GAPDH rabbit mAb, HRP-linked Antibody (Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit #9782 from Cell Signaling Technologies, MA, USA) and L-Dopa (#8786 Cell Signaling Technologies, MA, USA), Neuron-specific enolase (NSE) (#9536 Cell Signaling Technologies, MA, USA). Finally, immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence system (Amersham Biosciences, Freiburg, Germany).
9
Iris Compounds in EMT Regulation
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Irigenin and Tectorigenin used in this study were isolated from the rhizomes of Iris kashmiriana, as reported previously36 (link). Emodin, Safranal, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). DMEM, Fetal Bovine Serum (FBS), Penicillin and Streptomycin were obtained from Life Technologies Inc. (Gibco, USA). Epithelial-Mesenchymal Transition (EMT) antibody sampler kit was purchased from Cell Signaling Technology (Beverly, MA). Monoclonal antibodies against human Fibronectin EDA (IST-9) and β-actin were procured from Abcam and Sigma–Aldrich Co respectively.
10
Immunoblotting and Immunoprecipitation Techniques
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Western blot and immunoprecipitation were performed as described [25] . The primary antibodies included anti-Rad50 (Abcam, USA), anti-CARD9 (Protein Tech, USA), anti-p-p65 (Ser536), anti-p65, anti-p21 Cip1 , anti-p27 Kip1 , anti-Cyclin D1 (Cell Signaling Technology, USA),anti-MYC (Cell Signaling Technology, USA),anti-GAPDH(Zsbio, China) and anti-β-actin (Sigma-Aldrich, USA). Anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Twist and anti-Snail antibodies were all from the Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (Cell Signaling Technology, USA).
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