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5 protocols using β catenin

1

Western Blot Analysis of Protein Markers

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Protein lysates were acquired using RIPA lysis buffer supplemented with both phosphatase and protease inhibitor cocktails (Life Technologies, Thermo Fisher Scientific brand, Waltham, MA, USA). Equal amounts (5 μg) of proteins in RIPA buffer were separated on 10% NuPage Bis-Tris gels (Invitrogen) and transferred to a PVDF membrane (Invitrogen). Antibodies used are as follows: E-Cad (BD), PTP1B (BD, Fisher Scientific brand, Waltham, MA, USA), pSrc529 (Abcam), claudin-1 (Zymed, San Francisco, CA, USA), occludin (Zymed), β-catenin (Biolegend, San Diego, CA, USA), FAK (Cell Signaling, Danvers, MA, USA), pFAK (Cell Signaling) GAPDH (Abcam) and actin (Abcam) were used. In Figures 1,4 and 5, secondary antibodies were horsesadish peroxidase-conjugated anti-mouse or rabbit (BD Scientific; Fisher Scientific brand, Waltham, MA, USA) used at dilution 1:10 000. The protein bands were visualized using enhanced chemiluminescence (ECL) system (Thermo Scientific, Waltham, MA, USA). In Supplementary Figure 4, secondary antibodies were mouse or rabbit IRDye (Li-Cor, Lincoln, NE, USA) used at 1:10 000 and protein bands were detected using the Odyssey Infrared Imaging System (Li-Cor) and then fluorescent images were converted to gray scale.
All western blots are representative of three separate experiments.
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2

Protein Expression Analysis by Western Blot

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Proteins in cells were resolved in lysis buffer which supplemented with 1 mM PMSF, and lysed for 15 min on ice. Briefly, collected the supernatant after centrifugation at 12,000×g for 10 min and diluted in 5×SDS-PAGE loading buffer (Biolegend, San Diego, CA), heated at 95 °C for 5 min and cooled on ice. Protein concentration of each sample was quantified by using of the BCA assay. Then these proteins were transferred onto the PVDF membrane (Bio-Rad, Hercules, CA) after separated. Then the membranes were blocked and reacted with first antibodies against CtBP2, E-cadherin, β-catenin, Vimentin (Biolegend, San Diego, CA) and β-actin (Abcam, Cambridge, MA) in a 4 °C refrigerator overnight. The β-actin was used for the purpose of an internal control. Following, the membranes were reacted with secondary HRP-conjugated antibody. The protein bands were visualized and imaged via chemiluminescence detection system (Tanon, Shanghai, China), and quantified by ImageQuant TL (GE healthcare life sciences, Pittsburgh, PA).
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3

Immunocytochemistry for sFRP4, H2AX, β-catenin

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Cells were subjected to treatment and immunocytochemistry was performed as previously described using sFRP4 (Abcam, Cambridge, MA, USA), H2AX (Cell Signaling Technology, Beverly, MA, USA), and β-catenin (BioLegend, San Diego, CA, USA) [11 (link)].
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4

Western Blot Analysis of Signaling Proteins

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Protein expression levels of sFRP4 (Invitrogen), β-catenin (BioLegend), GSK-3β (Cell Signaling Technology), and APAF1 (R&D Systems, Minneapolis, MN, USA) were analyzed by Western blotting as previously described by Warrier et al. [12 (link)].
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5

Western Blot Analysis of Cell Adhesion Proteins

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Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 10% skim milk and incubated with the antibodies against E-cadherin (BioLegend), β-catenin (BioLegend), claudin-1 (Santa Cruz Biotechnology), PARD3 (Merck Millipore), Akt (Cell Signaling Technology), phospho-Akt (Ser473) (Cell Signaling Technology), PLCδ1 (Santa Cruz Biotechnology), N-cadherin (BD Biosciences), and β-actin (Sigma-Aldrich), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). Images were captured using LuminoGraph I (ATTO, Tokyo, Japan).
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