Equal amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Bovine serum albumin (5%) or skimmed milk was performed to block membranes. Primary antibodies include Fzd2 and CD44 (1:5,000; Abcam); interleukin-6 (IL-6), Stat3, p-Stat3 (Tyr705), non-phospho β-catenin, E-cadherin, vimentin, Slug, Wnt5a/b, Yes-associated protein 1 (Yap1), TGF-β1, Smad3, and ABCG2 (1:1,000; Cell Signaling Technology, Boston, MA, USA); Wnt3, Col1a1, and Col6a1 (1:1,000; Abcam); Zeb1 (1:500; Sigma-Aldrich, St. Louis, MO, USA); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:500; Proteintech, Chicago, IL, USA) were incubated with the membranes overnight. SuperSignal Chemiluminescent Substrates (Thermo Fisher Scientific) and imaging systems were used to analyze the results.
Wnt5a b
Manufactured by Cell Signaling Technology
Sourced in United States
Wnt5a/b is a recombinant human protein produced in HEK293 cells. It is a member of the Wnt family of secreted signaling proteins.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (8 mentions)
β catenin, by Cell Signaling Technology (8 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Vimentin, by Cell Signaling Technology (5 mentions)
N cadherin, by Cell Signaling Technology (5 mentions)
Snail, by Cell Signaling Technology (4 mentions)
Wnt3a, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Axin1, by Cell Signaling Technology (3 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Smad3, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Phospho lrp6, by Cell Signaling Technology (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
P sapk jnk, by Cell Signaling Technology (2 mentions)
Lamin a c, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Non phospho β catenin, by Cell Signaling Technology (1 mentions)
Tgf β1, by Cell Signaling Technology (1 mentions)
19 protocols using wnt5a b
1
Protein Expression Analysis by SDS-PAGE
2
Quantifying Wnt Pathway Proteins
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Total protein was isolated from CS-1 and SW1353 cells with 1 × RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). Protein concentrations were examined by Protein Assay Reagents (Bio-Rad, Hercules, USA). Equal amounts of denatured proteins were separated by NuPAGE® 4–12% Bis-Tris Gel (Life Technologies) and transferred onto a nitrocellulose membrane (Bio-Rad). After blocking in 5% non-fat milk for two hours, the membranes were incubated with specific primary antibodies WIF1 (Cell Signaling Technology, catalog number: 5652, 1:1000 dilution), Wnt5a/b (Cell Signaling Technology, catalog number: 2530, 1:1000 dilution), LRP6 (Cell Signaling Technology, catalog number: 3395, 1:1000 dilution), Dvl2 (Cell Signaling Technology, catalog number: 3224, 1:1000 dilution), and β-Actin (Sigma-Aldrich, dilution 1:2000) at 4 °C overnight. The membranes were further probed with respective secondary antibodies (LI-COR Biosciences, NE, USA), and scanned by Odyssey® CLx equipment (LI-COR Biosciences, NE, USA) to detect the bands and the density.
3
Bone Morphogenetic Pathway Signaling Assay
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The Human Phospho-Kinase Array Kit and recombinant human BMP-2/ BMP-9 were purchased from R&D Systems (Minneapolis, MN, USA). The primary antibodies against anti-human Phospho-p53 (Ser6, 9, 15, 46, 392, and Thr81), Wnt5a/b, LRP6, Phospho-LRP6, Dvl2, Dvl3, Axin1, GSK3β, Phospho-GSK3β, PI3K, Phospho-PI3K, AKT, Phospho-AKT, MDM2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). The iScript cDNA Synthesis Kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All primers and probes (RUNX2 #Hs00231692_m1; ALPL #Hs010291444_m1; BGLAP #Hs01587814_g1; NOG #Hs00271352_s1; GAPDH #Hs99999905-m1) were purchased from Applied Biosystems, Inc. (Bedford, MA, USA). Moreover, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal Master mix were from Applied Biosystems, Inc.
4
Signaling Pathway Antibody Analysis
5
Quantitative Protein Expression Analysis
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Primary antibodies N-cadherin, E-cadherin, Vimentin, β-catenin, p-GSK3β (S9), Dvl-2, Wnt5a/b, Notch1, CD44, p-Akt (ser473), Snail, and actin were purchased from Cell Signaling Technology (CST; Danvers, MA). CD44, CD133, and CD24 antibodies were purchased from Biolegend (BioLegend, Inc., San Diego, USA). Secondary Alexa 488-conjugated antibody and HRP conjugated antibody were purchased from CST.
6
Western Blot Analysis of Signaling Proteins
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Cells for western blotting were cultured in RPMI 1640 medium with 10% FBS for 24 h. Protein expression was detected with rabbit antibodies: p62/IMP2, β-catenin, Wnt3a, Wnt 5a/b, GSK3β, Snail, N-cadherin, and GAPDH (Cell Signaling Technology, Danvers, MA, USA); and mouse antibodies: E-cadherin (Cell Signaling Technology, Danvers, MA, USA). Primary antibodies were detected with goat anti-mouse IgG (HPR conjugate) or anti-rabbit IgG (HPR conjugate). The membranes were finally scanned by Invitrogen iBright Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
7
Immunoblotting Analysis of Cell Signaling Proteins
8
β-catenin and GPC5 Expression Analysis
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As previous described, the expression alteration of β-catenin and GPC5 was both detected by Immunofluorescence [12 (link)] and western blot [6 (link)]. The β-catenin was labelled with fluorescein isothiocyanate (FITC) and the GPC5 was labeled with tetramethyl rhodamine isothiocyanate (TRITC). WNT/β-catenin signaling pathway-related proteins (wnt3a, wnt5a/b, cyclinD1, MMP2, MMP7, MMP9, E-cadherin, β-catenin) and internal control proteins (LaminB, β-actin, Gapdh) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Protein density on scanned western blots was analyzed using Image J 1.44 software, and we performed semi-quantitative analysis for the results of the western blot analysis.
9
Molecular Signaling Pathways in Metabolic Regulation
10
Immunoblotting Workflow for Protein Analysis
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