Min6 cells were divided into groups and cultured alone, with ISC cell supernatant, or with exogenous Wnt5a (0.05 μg/ml) for 48 h. After 48 h, western blotting was performed [11 (link)] with the primary antibodies specific for the following proteins: Wnt5a (1 : 3000, Abcam, UK), β-catenin (1 : 1000, Proteintech, China), receptor tyrosine kinase-like orphan receptor 2 (Ror2) (1 : 1000, Santa Cruz, USA), phosphorylated Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CamKII) (1 : 2000, Abcam, UK), FoxO1 (1 : 3000, Abcam, UK), pFoxO1 (1 : 3000, Abcam, UK), PDX1 (1 : 3000, Abcam, UK), Glut2 (1 : 3000, Abcam, UK), CASK (1 : 2000, Abcam, UK), and GAPDH (1 : 5000, Sigma, USA).
Wnt5a
Manufactured by Abcam
Sourced in United States, United Kingdom
Wnt5a is a secreted signaling protein that belongs to the Wnt family. It is involved in various cellular processes, including cell fate determination, cell migration, and embryonic development.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by Abcam (4 mentions)
Wnt3a, by Abcam (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Camkii, by Proteintech (3 mentions)
Erk1 2, by Proteintech (3 mentions)
Gapdh, by Proteintech (3 mentions)
α tubulin, by Proteintech (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Gapdh, by Abcam (3 mentions)
Il 10, by Abcam (3 mentions)
Cd163, by Abcam (3 mentions)
β actin, by Abcam (3 mentions)
Wnt7b, by Abcam (3 mentions)
β actin, by Merck Group (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Phosphatase inhibitor, by Aspen (2 mentions)
Ripa lysis reagent, by Aspen (2 mentions)
Bca protein assay reagent kit, by Aspen (2 mentions)
Sds loading buffer, by Aspen (2 mentions)
Ecl developer, by Aspen (2 mentions)
32 protocols using wnt5a
1
Investigating Wnt5a Signaling in Pancreatic Beta Cells
2
Western Blot Analysis of rMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
rMSCs were lysed with RIPA buffer containing protease and phosphatase inhibitors (Biyotime). The protein concentration was measured by a BCA Protein Assay kit (Biyotime). Equal amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane, blocked in 5% milk, and immunoblotted with primary antibodies overnight at 4°C. The membranes were washed in TBST and incubated with a corresponding secondary antibody for 1h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Pierce). The following primary antibodies were used: Wnt5a (1:300, Abcam) and β-actin (1:1,000,CST).
3
Tissue Microarray Analysis of ROR2 and Wnt5a
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NSCLC and matched, tumor-adjacent tissues were prepared and used for TMAs. The TMAs were assembled using a tissue arraying instrument (Quick-Ray, UT06; UNITMA, Korea). Core tissue samples (2 mm in diameter) were taken from individual paraffin-embedded sections and deposited in recipient paraffin blocks. TMA specimens were cut into 4-μm sections and put on the super frost-charged glass microscope slides before immunohistochemical processing. Immunohistochemical analysis was performed as previously described. The slides were incubated with the primary antibodies against ROR2 (1:100; LS-C99125, LifeSpan BioSciences, Seattle, WA, USA) or Wnt5a (1:200; Abcam, Cambridge, MA, USA) at 4°C overnight. Horseradish-peroxidase-conjugated rabbit IgG (Abcam) was applied as the secondary antibody for ROR2, and anti-mouse IgG (Abcam) was applied for Wnt5a. The binding of the primary antibody was detected using diaminobenzidine solution. Slide in which primary antibody was omitted was used a negative control, while a breast cancer sample known to be ROR2 positive was included as a positive control.
4
Comprehensive Antibody Panel for Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used in this study were as follows: EGFR (1:50; # ab52894), HER3 (1:25; # ab5470), HER4 (1:150; # ab19391), VEGFR3 (1:100; # ab27278), VEGF-C (1:50; # ab135506), Wnt5a (1:100; # ab72583), Beta-Catenin (1:100; # ab32572), p-Akt1 (1:100; # ab32505), Akt1 (1:50; #ab59380) were purchased from Abcam company, UK. HER2 (1:100, #4290) was purchased from Cell Signaling Technology, Inc., USA. EphrinA1 (1:100; # sc-911) and EphA3 (1:100; # sc-920) were purchased from Santa Cruz Biotechnology, USA. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako EnVision, USA).
5
Comprehensive Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed according to the previous report 8 (link). Briefly, total proteins were separated by 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% skim milk, the PVDF membrane was incubated with primary antibodies and HRP-conjugated secondary antibodies. The primary antibodies are as follows: Wnt5a (1:1000, Abcam), p-ERK1/2 (1:1000, CST), ERK1/2 (1:1000, Protein Tech), p-CaMKII (1:1000, CST), CaMKII (1:1000, Protein Tech), α-Tubulin (1:5000, Protein Tech), GAPDH (1:5000, Protein Tech).
6
Immunohistochemical Analysis of Wnt5a and Fzd5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
7
Comprehensive Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed using previously described protocols. After the concentrations of protein were measured via BCA (Aspen), the protein was separated into equal amounts via SDS-PAGE (Beyotime Biotechnology, Shanghai, China), transferred into the PVDF membrane (Millipore, Burlington, MA, USA) and then incubated with 5% bovine serum albumin for 1 h at 25 °C. Next, the membranes were incubated overnight with primary antibodies specific for CD9 (ABCAm), CD63 (ABCAm), TSG101 (ABCAm), Runx2 (ABCAm), collagen I (ABCAm), OCN (Santa), AKT (CST), ERK (CST), JNK (CST), p-65 (CST), β-catenin (CST), Wnt3a (ABCAm), Wnt5a (ABCAm), Wnt8a (Bioss), Wnt10b (Isbio) and GAPDH (ABCAm). Then, samples were mixed with the secondary antibodies (1:2,000, 30 min). The membranes were incubated with Immobilon ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the bands were quantified via software.
8
Western Blot Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100 °C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4 °C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed five times for 30 min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 (1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII (1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).
9
Immunohistochemical Analysis of Wnt/β-Catenin Pathway in Pancreatic Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin blocks with tumor samples were obtained from 73 patients who had undergone curative resection for pancreatic cancer between January, 2001 and December, 2009. Tissue microarray was constructed as mentioned previously [10 (link)]. We performed immunohistochemical staining with the following primary antibodies at specified dilutions: Wnt-3a (Abcam) diluted 1:100, Wnt-5a (Abcam) diluted 1:50, and β-catenin (Abcam) diluted 1:100. The results were interpreted by two independent pathologists who were blinded to the specific diagnosis and prognosis of each case. The staining intensity was scored on a three-tiered scale: score 0, less than 10% of cells positive; 1, 10% to 49% positive; and 2, more than 50% of cells positive. We also reviewed the patients’ medical records to analyze the clinical outcome according to Wnt/β-catenin expression. This study was approved by our Institutional Review of Board: Board in Seoul St. Mary's Hospital (KC11SISI0184).
10
Proteomic Analysis of HANP Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SK-MEL-28 cells were seeded in 60 mm culture dishes at a density of 6 × 105 cells/dish with 240 µg/ml HANPs and without HANPs. Each group contained three parallel samples. One day later, cells were ground with liquid nitrogen and peptides were processed according to the manufacturer’s protocol for the tandem mass tag (TMT) kit (Thermo Scientific, USA). The differentially expressed proteins (DEPs) were satisfied with the following conditions: unique peptides ≥1 with average ratio-fold change ≥1.5 (up-regulation) and ≤0.67 (down-regulation), as well as P values ≤ 0.05. Furthermore, the DEPs were analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The pathway with a corrected P value < 0.05 was considered significant. As mentioned in Cell apoptosis part, insulin-like growth factor binding protein 3 (IGFBP3) (1:1000), IGFBP5 (1:1000), thrombospondin 1 (THBS1) (1:1000), apolipoprotein A1 (APOA1) (1:1000), Dickkopf 1 (DKK1) (1:1000) and Wnt5a (1:1000) which were purchased from Abcam (USA) were analyzed by WB analysis.
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!