Cells (HPDE6-C7, CaPAN-1, BxPC-3, PANC-1, and AsPC1) were lysed via RIPA buffer (Beyotime, Shanghai, China) and protein was collected. Concentrations of total protein were quantified through a BCA Protein Assay Kit (Sigma-Aldrich). Equal amounts of each sample were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmembrane to polyvinylidene fluoride (PVDF) membranes (Merck, Darmstadt, Germany). Immediately, the membranes were incubated with primary antibodies overnight at 4°C, which were FBXL7 (ab59149, Abcam; 1.25 μg/ml), E-Cadherin (ab231303, Abcam; 1 μg/ml), N-cadherin (ab18203, Abcam; 1 μg/ml), Vimentin (ab137321, Abcam; 1:3,000), Snail 1 (ab53519, Abcam; 3 μg/ml), Snail 2 (ab82846, Abcam; 1:500), ZEB 1 (ab155249, Abcam; 1:3,000), Twist (ab49254, Abcam; 2.5 μg/ml), and GAPDH (ab181602, Abcam; 1:10,000). Goat anti-Human IgG/HRP (Solarbio, SE101) was employed as secondary antibody to incubate the membranes. Finally, Positive signals were detected with an ECL luminescence reagent (Sangon Biotech, Shanghai, China).
Ab155249
Manufactured by Abcam
Sourced in United Kingdom
Ab155249 is a lab equipment product from Abcam. It is a laboratory tool designed for a specific function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Ab53519, by Abcam (1 mentions)
Ab49254, by Abcam (1 mentions)
Ab137321, by Abcam (1 mentions)
Ab59149, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Ab82846, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ecl luminescence reagent, by Sangon (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab8978, by Abcam (1 mentions)
Ab6302, by Abcam (1 mentions)
Chip kit, by Merck Group (1 mentions)
Ab187008, by Abcam (1 mentions)
Ab180714, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab180714, by Abcam (1 mentions)
4 protocols using ab155249
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of EMT Markers
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The cells were harvested, washed with PBS and extracted with lysis buffer (150 mM NaCl, 1 mg/mL leupeptin, 1 mM EDTA, 1 mM EGTA, 1 mM natrium glycerophosphoricum, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM DTT and 1×cocktail). After the cell debris were removed by centrifugation at 14,000 g for 10 min at 4°C, the protein concentration was measured by Nanodrop. Then the proteins were separated using 10% SDS-PAGE and transferred onto a nitrocellulose filter membrane. To block nonspecific binding, the membranes were incubated with 5% non-fat milk for 2 hours at room temperature. After the membranes were incubated overnight at 4°C with the human anti-PFTK1 (Ab175489; Abcam, UK), anti-ZEB1 (Ab155249; Abcam, UK), anti-vimentin (Ab8978; Abcam, UK) and anti-β-catenin (Ab6302; Abcam, UK), appropriate secondary antibodies conjugated to horseradish peroxidase were diluted 1:3000 and incubated with the membrane. GAPDH was used as an internal reference. After washing three times with TBST, proteins were detected using ECL reagents (Pierce, USA) and exposed to Alpha Innotech Imager.
3
ChIP-qPCR Analysis of ZEB1
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ChIP analysis was performed using a ChIP kit (Millipore). The cells were fixed using 4% paraformaldehyde for 10 min at room temperature, and DNA was sonicated until the sheared DNA was ~ 200–1000 bp in size. The supernatant was harvested by centrifugation at 13,000 rpm for 5 min at 4 °C, and the chromatin was incubated overnight with the antibody against ZEB1 (ab155249, Abcam). After immunoprecipitation of the cell lysates overnight, qPCR was used to analyze the enrichment of the POLDIP2 promoter.
4
Epithelial-Mesenchymal Transition Protein Analysis
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Cultured cell lysates were prepared using RIPA buffer and Phenylmethanesulfonyl fluoride (PMSF) (both from Beyotime, Nantong, China) and then separated on 10% Sodium dodecyl sulfate (SDS) polyacrylamide gels. Primary antibodies against Snail (Abcam, #ab180714), FBP1 (Abcam, #ab180714), E-cadherin (Cell Signaling Technology, #9782), Vimentin (Cell Signaling Technology, #9782), Slug (Cell Signaling Technology, #9782), zinc-finger E-box-binding homeobox 1 (ZEB1; Abcam, #ab155249) and Twist1 (Abcam, #ab187008) were used. Protein levels were normalised against β-tubulin.
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