Radioimmunoprecipitation assay buffer (Beyotime, Nantong, China) containing protease and phosphatase inhibitors was used to cleave tissues and cells and extract proteins, with electrophoresis transfer membrane sealing, incubation, washing, and other processes also being conducted. PORCN (ab201793, 1:1,000, Abcam, Cambridge, UK), β-catenin (GB12015, 1:1,000, Servicebio, Wuhan, China), E-cadherin [3195T, 1:1,000, Cell Signaling Technology (CST), Danvers, MA, USA], N-cadherin (13116T, 1:1,000, CST), and α-smooth muscle actin (SMA) (ab5694, 1:10,000, Abcam) antibodies were used in this study. The trend of the protein band was quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Porcn
Manufactured by Abcam
Sourced in United Kingdom
PORCN is a lab equipment product. It is a protein that plays a role in the post-translational modification of Wnt proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
β catenin, by Wuhan Servicebio Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Pakt ser473 antibody, by Cell Signaling Technology (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
2 protocols using porcn
1
Western Blot Analysis of EMT Markers
2
Evaluating WNT and PI3K Pathway Modulation
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Target modulation was assessed using qPCR for Axin2 (a marker of WNT974), and western blot for porcupine and phospho-Akt Ser 473 (a marker of buparlisib activity). qPCR was performed using Taqman assays (Life Technologies). Protein was isolated after 48 h of treatment in biological triplicates using radioimmunoprecipitation assay buffer and quantified with bicinchoninic acid assay (Thermo Fisher). Protein was run on bis-tris gels (Life Technologies) and transferred to polyvinylidene fluoride membrane. Primary pAKT-Ser 473 antibody (Cell Signaling Technology, Beverly, Massachusetts), β-actin (Cell Signaling Technology), and PORCN (Abcam, Cambridge, England) was incubated overnight with subsequent 1-hour incubation with horseradish peroxidase-conjugate secondary antibody. Staining was performed using a chemiluminescent substrate (ThermoFisher). Imaging was performed using an LAS-4000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). Quantification of blots was performed using ImageJ (NIH) and statistical analysis using GraphPad.
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