For immunological detection, 20 μg of cell lysate was separated using SDS-PAGE (7.5–10% Tris gel). Then, the proteins were blotted onto a PVDF membrane, which was incubated with the PVDF blocking reagent Can Get Signal (TOYOBO, Osaka, Japan). Proteins were probed with primary antibodies against β-catenin (1/1000, Abcam, Cambridge, MA, USA), FOXF2 (1/1000, Abnova, Walnut, CA, USA), WNT2B (1/2000, Abcam), LMNB1 (1/2000, Proteintech, Rosemont, IL, USA), and GAPDH (1/2000, MBL, Aichi, Japan). A horseradish peroxidase-conjugated goat anti-rabbit antibody was then added, and secondary antibodies were detected through autoradiography using enhanced chemiluminescence (ECL Plus, General Electric Healthcare, Chicago, IL, USA).
Wnt2b
Manufactured by Abcam
Sourced in United States, United Kingdom
WNT2B is a protein that is a member of the Wnt family. It plays a role in cell signaling pathways involved in cell fate determination and patterning during embryonic development. The specific functions and applications of this protein are not provided in order to maintain an unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
C myc, by Abcam (3 mentions)
β catenin, by Abcam (3 mentions)
β actin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
Runx2, by Cell Signaling Technology (2 mentions)
Lmnb1, by Proteintech (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Can get signal, by Toyobo (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Active β catenin, by Abcam (1 mentions)
Total β catenin, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Anti mouse epcam, by BioLegend (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
8 protocols using wnt2b
1
Immunoblotting of Cell Lysate Proteins
2
Western Blot Analysis of Wnt/β-catenin Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The U2OS and MG63 cells after different treatments were lysed in RIPA buffer supplemented with 1% protease inhibitors (Sigma). Protein concentrations were measured by BCA assay kit, and the protein samples were then denatured at 98°C for 10 min. Equal amounts of the denatured proteins were then subjected to electrophoresis on a 10% SDS-PAGE gel followed by transferring to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). The PVDF membranes were incubated with 5% skimmed milk for 1 hrs at room temperature. Subsequently, the PVDF membranes were incubated with different primary antibodies against WNT2B (Abcam, Cambridge, USA), active β-catenin (Abcam), total β-catenin (Abcam), cyclin D1 (Abcam), c-myc (Abcam) and β-actin (Abcam) at 4°C overnight. After washing with Tris-buffered saline containing 0.1% Tween 20 for 3 times, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam) for 2 hrs at room temperature. The protein bands were detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and β-actin was used as the internal control.
3
Immunohistochemical Analysis of Intestine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hematoxylin and eosin staining (Muto Pure Chemicals, Tokyo, Japan) and immunohistochemical analysis were performed as previously described51 (link). For immunohistochemistry, samples of large intestine were fixed in 4% paraformaldehyde (Wako) and incubated in 20% sucrose. The tissues were embedded in OCT compound (Sakura Fine Technical Company, Tokyo, Japan). Tissue sections were stained with anti-mouse EpCAM (Biolegend, San Diego, CA, USA), anti-mouse gp38 (Biolegend), Ki67 (Biolegend), and Wnt2b (Abcam). Anti-rabbit IgG conjugated with Alexa 488 (Thermo Fisher Scientific) served as a secondary antibody.
4
Depletion of Wnt proteins from conditioned media
5
Protein Expression Analysis in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from 1 × 106 cells on six‐well plates using RIPA buffer (Beyotime). Every 1 ml lysate was added with 10μg phenylmethylsulfonyl fluoride (PMSF), and the concentration of protein was quantified using BCA working solution (Beyotime). Proteins were electrophoresed in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes, followed by sealing in 5% defatted milk. The membrane was incubated with the primary antibodies against matrix metallopeptidase 2 (MMP2; Abcam, 1/1000), matrix metallopeptidase 9 (MMP9; Abcam, 1/1000), E‐cadherin (Abcam, 1/1000), N‐cadherin (Abcam, 1/1000), WNT2B (Abcam, 1/1000), lymphoid enhancer binding factor 1 (LEF1; Abcam), β‐catenin (Abcam), c‐MYC (Abcam), cyclin D1 (CCND1; Abcam), cyclin dependent kinase 4 (CDK4; Abcam), Snail (Abcam) and GAPDH (Abcam, 1/1000) for detection of specific proteins. Then the membrane was cultured with HRP‐labeled secondary antibody. The protein bands were measured using enhanced chemiluminescence (ECL) western blotting substrate (Millipore). The original images were demonstrated in Supplementary file 2–3.
6
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the cells by lysis with radioimmunoprecipitation assay buffer (DGCS Biotechnology, China). The protein content of the lysate was then determined using the BCA Protein Assay Kit (Beyotime, China) according to the manufacturer’s protocol. Cell lysates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membrane was blocked with TBS-T (0.1% Tween-20 in TBS) containing 5% BSA, and incubated with primary antibody overnight at 4 °C. After three washes with TBS-T, the membrane was incubated with HRP-conjugated secondary antibody for 1 h and then washed with TBS-T. Immune complexes were detected by chemiluminescence (SuperSignal West Femto; Pierce Biotechnology, Waltham, MA) using a MyECL Imager (Thermo Scientific, Waltham, MA). Mean densitometry data from independent experiments were normalized to the control. Primary antibodies are listed as follows: Runx2 (1:1000, Cell Signaling Technology, Danvers, MA), GAPDH (1:1000, Cell Signaling Technology), WNT2B (1:800, Abcam, Cambridge, UK), osteocalcin (OCN) (1:1000, Abcam, Cambridge, UK), ALP (11,000, Abcam, Cambridge, UK). All experiments were performed in triplicate.
7
Western Blot Antibody Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for western blot: c-Myc (Abcam, ab32072, 1:1000), Wnt2b (Abcam, ab178418, 1:1000), beta Catenin (Abcam, ab68183, 1:1000), E-cadherin (Abcam, ab40772, 1:1000), α-catenin (Abcam, ab51032, 1:1000), N-cadherin (Abcam, ab76011, 1:1000), Vimentin (Abcam, ab92547, 1:1000), β-actin (Abcam, ab8226, 1:1000), Goat anti mouse second antibody (Cell Signaling Technology, 5470S, 1:15000), Goat anti rabbit second antibody (Cell Signaling Technology, 5151S, 1:30000).
8
Protein Expression Analysis of BMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins of BMSCs or bone tissues were extracted using the Total Protein Extraction Kit (Signalway Antibody LLC, Maryland, USA) according to the manufacturer's instructions. SDS-PAGE gels were utilized to separate proteins, which were then transferred onto PVDF membranes. Next, the PVDF membranes were incubated with the primary antibodies against β-catenin (Santa Cruz Biotechnology, USA; sc-7963; 1:1000), Wnt2b (Abcam, UK; ab178418; 1:1000), ALP (1:1000, Santa Cruz Biotechnology, USA), Runx2 (Cell Signaling Technology, USA; 12556; 1:1000), Ocn ( Santa Cruz Biotechnology, USA; sc-390877; 1:1000), Opn (Santa Cruz Biotechnology, USA; sc-21742; 1:1000), Lamin B1 (Abcam, UK; ab16048; 1:5000), and, Gapdh (Abcam, UK; ab9485; 1:5000) overnight at 4 °C, after which the corresponding secondary antibodies were applied for 1 h at room temperature. Finally, RapidStep™ ECL Reagent (Millipore, Germany) was used to visualize the protein bands, with GAPDH used as the loading control. Finally, RapidStep™ ECL Reagent (Millipore, Merck KGaA, Darmstadt, Germany) was used to visualize the protein bands.
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!