Whole Pancreatic cancer cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland) after culturing in MRC-5-CM for 14 days. Western-blot analysis was performed by established protocols 43 (link). Anti-CTNNA1 and CTNNB1, anti-E-cadherin, anti-N-cadherin, anti-ZEB-1, ZEB-2, Snail, Slug and anti-vimentin primary antibodies were purchased from (Abcam); anti-MMP-2, 3, 13 and 21, anti-WNT-2, anti-WNT16, anti-TGFB1, anti-Src, P-Src-Y418 and P-Src-Y529 primary antibodies were purchased from (Epitomics); anti-Bcl-2, Bcl-xl, Bad, Bax, Bim, Bid, Caspase-3 and anti-β-actin were from ( Cell Signaling Technology).
Anti wnt 2
Manufactured by Abcam
Sourced in China, United Kingdom
Anti-WNT-2 is a laboratory reagent used for research purposes. It is an antibody that specifically binds to the WNT-2 protein, which is a member of the Wnt family of secreted glycoproteins. The core function of Anti-WNT-2 is to enable the detection and analysis of WNT-2 in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor mixture cocktail, by Roche (3 mentions)
Anti wnt16, by Abcam (3 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti β catenin, by Abcam (3 mentions)
Anti zeb1, by Abcam (2 mentions)
Anti tgfb1, by Abcam (2 mentions)
Anti src, by Abcam (2 mentions)
P src y418, by Abcam (2 mentions)
P src y529, by Abcam (2 mentions)
Snail, by Abcam (2 mentions)
Anti e cadherin, by Abcam (2 mentions)
Anti wnt5b, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti n cadherin, by Abcam (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bca kit, by Beyotime (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Abcam (1 mentions)
5 protocols using anti wnt 2
1
Molecular Mechanisms of Pancreatic Cancer Progression
2
Molecular Profiling of Epithelial-Mesenchymal Transition
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Experimental cancer cells (after culturing in HUVEC-CM for 21 days) and respective control cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). Western-blot analysis was performed by established protocols 12 . Anti-α-catenin, anti-E-cadherin, anti-ZEB-1, ZEB-2, Snail, Slug, anti-ZO-1 and anti-Laminin A1 primary antibodies were purchased from (Abcam); anti-MMP-1, -2, -3, -11, -12, -13, -17 and -21, anti- ITGA6, B1, B3, B4, B7, anti-FAK, P-FAK-Y397, anti-Src, P-Src-Y418, P-Src-Y529, anti-fibronectin, anti-Laminin B3, anti-Wnt-2, anti-Wnt-5B, anti-Wnt-16, anti-TGF-β, anti-β-catenin and anti-N-cadherin primary antibodies were purchased from (Epitomics); anti-Cdc-2, P-Cdc-2-Tyr15, CDK4, CDK6, Cyclin A, Cyclin D1, Cyclin D3, Cyclin E2, P15, P16, P21, P27, P53, Rb, P-Rb-S811, P-Rb-S780; anti-Bcl-xl, Bad, P-Bad-Ser112, Bak, Mcl-1, Puma and anti-β-actin were from (Cell Signaling Technology).
3
Western Blot Analysis of Liver Cancer Cells
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Following culture in MRC-5-CM for 14 days, whole liver cancer cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). After centrifugation at 12000rpm for 30 minutes at 4℃, the protein concentrations of supernatants in samples were measured by the BCA protein assay (Thermo scientific, Rockford, IL, USA). Equal amounts of protein (50μg) were separated by 10%-12% NUPAGE Bis-tris Gel (Invitrogen, CA, USA) electrophoresis (constant voltage: 120mv) and transferred onto polyvinylidene fluoride (PVDF, 0.45μm) membranes (constant current: 350mA for 70/120 min). After being blocked by Tris-buffered saline and Tween 20 (TBST) buffer containing 5% non-fat powder milk for 2h, the membranes were incubated with primary antibodies overnight on ice. After washing the membranes several times in TBST while agitating, detection was performed using the appropriate secondary HRP-conjugated anti-mouse or anti-rabbit antibody. Immunoreactive bands on the blots were visualized with enhanced chemiluminescence reagent ECL kit (Beit Haemek, Israel). Anti-GAPDH, anti-WNT-2, anti-WNT-5B, anti-WNT16, anti-TGFB1, anti-CTNNB1, anti-IL6, anti-Nanog, anti-OCT4 and anti-CK19 primary antibodies were purchased from (Epitomics).
4
Quantitative Protein Expression Analysis
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The total protein was extracted from the liver tissues using a Total Protein Extraction kit (Beyotime, Shanghai, China) and quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein per sample (30 μg) were separated on a 10% SDS–PAGE and electroblotted onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk for 2 h, the blots were incubated overnight with anti-VEGF (1:2000; Wanleibio, Shenyang, Liaoning, China), anti-Cyclin D1 (1:1000; Wanleibio, Shenyang, Liaoning, China), anti-NLRP3 (1:2000; Bioss, Beijing, China), anti-Wnt2 (1:1000; Abcam, Waltham, MA, USA), anti-β-catenin (1:10,000; Abcam, Waltham, MA, USA), anti-P-P65 (1:500; Santa, Dallas, TX, USA), anti-P65 (1:3000; Proteintech, Wuhan, Hubei, China), anti-caspase-1 (1:500; Santa, Dallas, TX, USA), anti-ASC (1:500; Santa, Dallas, TX, USA), anti-GSDMD (1:2000; Affinity, Liyang, Jiangsu, China), and anti-β-tubulin (1:1000; Wanleibio, Shenyang, Liaoning, China) primary antibodies at 4 ℃. The blots were washed with TBST and incubated with HRP-conjugated anti-IgG for 2 h. The positive bands were detected using an enhanced ECL reagent (Meilunbio, Dalian, Liaoning, China) on an AI600 System (GE Healthcare, Pollards Wood, UK). The relative protein expression was quantified using ImageJ software and normalized against the β-tubulin control.
5
Apoptosis and Wnt Signaling Pathway Analysis
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Protein samples were collected from cell lysates and protein concentrations were determined using a BCA kit (Beyotime). Proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% Tris-buffered saline with Tween 20 for 2 hours at room temperature and then incubated with primary antibody overnight at 4°C. The primary antibodies were: anti-caspase-3, anti-cleaved caspase-3, anti-caspase-9, anti-cleaved caspase-9, anti-BAX, anti-Bcl-2, anti-CytC, anti-Apaf-1, anti-COX IV, anti-PAX2, anti-GSK-3β, anti-pGSK-3β(Ser9), anti-β-catenin, anti-p-β-catenin(Ser33+Ser37), anti-Wnt2, anti-Wnt4, anti-Wnt5a, anti-Wnt10b, anti-Wnt11, anti-Wnt13, anti-Wnt14 (Abcam, Cambridge, UK), anti-GAPDH, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA). Films were cleaned 3 times with TBST and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 hour. Membranes were visualised using the enhanced chemiluminescence reagents (Millipore, Burlington, MA, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
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