Total proteins were extracted using RIPA lysis buffer within 1% protease inhibitor cocktail (Millipore, Bedford, MA, USA) 0.1 mM according to the manufacturer’s instructions. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking, the membranes were then incubated at 4 °C overnight with the following primary antibodies: anti-HIF-1α, anti-VEGF-A, anti-JNK, anti-PPAR-γ, anti-C/EBP-α, anti-FABP4 (1:1000, all Cell Signaling Technology, Danvers, MA, USA), anti-NF-κB p65, anti-p-IκB, anti-TNF-α, anti-MCP-1 (1:1000, all Abcam, Cambridge, MA, USA), anti-SFRP5, anti-Wnt5a, anti-Wnt10b, anti-β-catenin (1:1000), and anti-β-actin (1:5000, all GeneTex, Irvine, CA, USA). After washing, the membranes were probed with corresponding second antibodies (1:3000, GeneTex). The density of the individual protein bands was quantified by densitometric scanning of the blots using ImageJ software.
Anti β catenin
Manufactured by GeneTex
Sourced in United States
Anti-β-catenin is a primary antibody that binds to the β-catenin protein. β-catenin is a key regulator of the Wnt signaling pathway and plays a role in cell-cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by GeneTex (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti pparγ, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti vegfa, by Cell Signaling Technology (1 mentions)
Anti c ebpα, by Cell Signaling Technology (1 mentions)
Anti fabp4, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Abcam (1 mentions)
Anti p iκb, by Abcam (1 mentions)
Anti mcp 1, by Abcam (1 mentions)
Anti wnt5a, by GeneTex (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Anti claudin 5, by Abcam (1 mentions)
Anti occludin, by Proteintech (1 mentions)
Anti adam17, by Abcam (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti β catenin
1
Western Blot Analysis of Adipogenic Markers
2
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To understand changes at the protein level, whole cell lysates prepared in radioimmunoprecipitation assay (RIPA) buffer were subjected to SDS-PAGE and western blotting, as described elsewhere. The following antibodies were used in this study: anti-claudin-5, anti-ADAM17 (Abcam, Cambridge, UK), anti-CD144, anti-β-catenin (Genetex, Irvine, CA), anti-occludin (Proteintech, Chicago, IL), anti-ZO-1 (Invitrogen, Waltham, MA), and anti-β-actin (Sigma-Aldrich, Chicago, IL). Densitometry of protein bands was performed using the Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA). β-actin was used as the loading control.
3
Immunohistochemical Staining of Tumor Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC staining of paraffin-embedded human or mice tumor sections were performed according to standard protocols. Sections were deparaffinized, rehydrated, subjected to antigen retrieval, and blocked with 3% hydrogen dioxide and goat serum, followed by incubating in primary antibodies overnight at 4 °C. First antibodies used were listed as follows: anti-CD36 antibody (1:200, Abcam), anti-β-catenin (1:200, Genetex), anti-cmyc (1:200, GeneTex), anti-HK2 (1:500, ABclonal), anti-PKM2 (1:500, ABclonal), anti-GLUT1 (1:250, Abcam), anti-LDHA (1:250, GeneTex), anti-GPC4 (1:200, Proteintech), anti-GFP (1:500, Proteintech), anti-Ki-67 (for mouse, 1:800, Cell Signaling), anti-Ki-67 (for human, 1:800, Cell Signaling), anti-PCNA (1:1000, Proteintech). Next day, the sections were put in room temperature for 30 min to rewarm, followed by secondary antibody incubation for 1 h in room temperature and DAB staining was performed with IHC assay kit (Maixin, China). Counterstaining was carried with hematoxylin for 2 min. Images were taken with OLYMPUS DP22 microscope. Antibodies were shown in Supplementary Table 3 .
4
Histological and Immunohistochemical Analysis of Colon Tissue in Colitis-Associated Colorectal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collected tissues from mice with CAC receiving AS1517499, 5-FU, Trymethyglycine, AS1517499+5-FU, and Trymetilglycine+5-FU, respectively, the untreated group with CAC (CAC), and finally the tissue obtained from healthy mice (Control), were fixed in 100% ethanol and embedded in paraffin for posterior 4-mm cross-sectioning. The tissue sections were stained with hematoxylin and eosin (H&E; for pathologic evaluation).
For immunohistochemical analysis, the sections were deparaffinized in xylene and then rehydrated with graded alcohols and processed as reported previously [69 (link)]. The sections were incubated overnight at 4 °C with the respective primary antibodies diluted in 1× PBS (anti-E-cadherin, 1:300, cell signaling; anti-β-catenin, 1:200, GeneTex, Irvine, CA, USA) and then developed following the conventional technique. The slides were analyzed using an AxioVert.A1 image capture optical microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany)Tissue microphotographs were captured using an AxioCam MRc and ZEN lite 2011 software v.1.0.1.0 (Oberkochen, Germany). Quantification of E-Cadherin and β-catenin was performed using ImageJ software v.4.9 by counting cells in 10 high-powered fields from each mouse.
For immunohistochemical analysis, the sections were deparaffinized in xylene and then rehydrated with graded alcohols and processed as reported previously [69 (link)]. The sections were incubated overnight at 4 °C with the respective primary antibodies diluted in 1× PBS (anti-E-cadherin, 1:300, cell signaling; anti-β-catenin, 1:200, GeneTex, Irvine, CA, USA) and then developed following the conventional technique. The slides were analyzed using an AxioVert.A1 image capture optical microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany)Tissue microphotographs were captured using an AxioCam MRc and ZEN lite 2011 software v.1.0.1.0 (Oberkochen, Germany). Quantification of E-Cadherin and β-catenin was performed using ImageJ software v.4.9 by counting cells in 10 high-powered fields from each mouse.
5
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS–PAGE and Western blot analysis were performed as described previously [24 (link)]. Briefly, cell lysates were prepared by vibrating cells in a RIPA buffer (150 mM Sodium Chloride, Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-Cl; pH 8.0). The protein concentration was determined with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). The immunoreactive bands were visualized via a light emitting non-radioactive method (ECL; Millipore). The sources of the antibodies used in western blot analyses are listed below: anti-γH2AX (Millipore; 05–636), anti-α-tubulin (Genetex, Irvine, CA, USA; GTX76511), anti-TRAX [25 (link)], anti-β-actin (Genetex; GTX11003), anti-DNA-PKCS Thr2609 (Abnova, Cambridge, United Kingdom; PAB10324), anti-DNA-PKCS (Abcam, Cambridge, United Kingdom; ab1832), anti-GSK3β Ser9 (Cell Signaling, Danvers, MA, USA; 9336), anti-GSK3β (Genetex; GTX83315), anti-β-catenin (Genetex; GTX61089), anti-DISC1 (Thermo; 710203), anti-A2AR (Santa cruz, Dallas, Texas, USA; SC-32261), and anti-PARP (Genetex; GTX112864).
6
Multimarker Profiling of Hepatic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were dissociated using dispase (Thermo Fisher Scientific) and a cell dissociation buffer (Thermo Fisher Scientific). Cells fixed with Cytofix Fixation Buffer (BD, USA) were washed with PhosFlow Perm/Wash Buffer I (BD) and incubated with primary antibodies (anti-HNF4α [GeneTex, USA], anti-CK19 [GeneTex], anti-AFP [R&D Systems, USA], anti-albumin [R&D Systems], anti-β-catenin [GeneTex], anti-Numb [R&D Systems], anti-Notch 1 ICD [R&D Systems], and anti-Hes5 [Bioss, USA] antibodies at a dilution of 1:400) at 4 °C overnight. After the cells were washed, they were incubated with Alexa 488 (Cell Signaling Technology, USA)- and phycoerythrin (Southern Biotech, USA)-conjugated anti-rabbit and anti-mouse IgG, respectively at 4 °C overnight. Then, the cells were washed again and passed through a 45-μm filter, and were subjected to flow cytometric analysis using a FACS Calibur (BD) flow cytometer according to the manufacturer's instructions. For flow cytometric analysis, the proportion of immunopositive cells was determined using the antibodies mentioned above. The data, which were normalized to the population of HNF4α+ cells, were represented as the relative value compared to that obtained following solvent control treatment.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!