Standard streptavidin-peroxidase immunohisto-chemistry procedures were performed following the manufacturer's instructions with an UltraSensitive TM SP Kit (Maixin-Bio, Fujian, China) as previously described [36 (link)]. Primary antibodies against PLCD1 (ab134936; Abcam), MMP7 (ab207299; Abcam), pERK1/2 (#4370; Cell Signaling Technology), or active-β-catenin (05-665; Millipore) were employed, and PBS was used as a negative control. All immunohistochemical images were subjected to mean optical density (OD) measurement using Image Pro Plus (IPP, version 6.0; Media Cybernetics, Silver Spring, MD, USA).
Active β catenin
Manufactured by Merck Group
Sourced in United States, China
Active β-catenin is a protein that plays a central role in the Wnt signaling pathway. It is an essential component of this cellular communication system that regulates various developmental and homeostatic processes. This lab equipment product allows for the detection and quantification of the active form of β-catenin.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by BD (7 mentions)
β catenin, by Merck Group (6 mentions)
Total β catenin, by BD (6 mentions)
E cadherin, by BD (6 mentions)
β actin, by Merck Group (6 mentions)
α tubulin, by Merck Group (5 mentions)
β catenin, by Cell Signaling Technology (5 mentions)
C myc, by Abcam (5 mentions)
Cyclin d1, by Cell Signaling Technology (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Axioskop 2 plus, by Zeiss (4 mentions)
Wnt3a, by Abcam (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Flag tag (flag), by Merck Group (3 mentions)
Gsk3β, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Axin1, by Cell Signaling Technology (3 mentions)
β catenin, by Santa Cruz Biotechnology (3 mentions)
Vimentin, by Abcam (3 mentions)
Total β catenin, by Cell Signaling Technology (3 mentions)
59 protocols using active β catenin
1
Immunohistochemical Analysis of Biomarkers
2
Investigating Wnt Signaling in EPSCs
3
Western Blot Analysis of Myogenesis
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C2C12 cells were harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Whole-cell protein extracts were quantified using the BCA assay, separated by SDS-PAGE 8–12%, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included Myogenin (Abcam, 1:1000, ab124800, Cambridge, MA, USA), MyoD (Santa Cruz, 1:200, sc-377460), HDAC9 (Abcam, 1:1000, ab59718), HIF1α (Abcam, 1:1000, ab179483), HIF2α (Abcam, 1:1000, ab179825), H3K9 (Abcam, 1:1000, ab32129), H3K14, H3K18, H4K16 (Cell Signaling, 1:1000), LC3I/II (Cell Signal, 1:1000, 12741), Beclin1 (Cell Signal, 1:1000, 3738), Atg5 (Cell Signal, 1:1000, 12994), Atg7 (Cell Signal, 1:1000, 8558), Atg12 (Cell Signal, 1:1000, 4180), p62 (Cell Signaling, 1:1000, 23214), p-GSK3β Ser9 (Cell Signal, 1:1000, 9323), GSK3β (Cell Signal, 1:1000, 12456), and active-β-catenin (Millipore, 1:800, 05–665). Stripped membranes were reprobed with GAPDH (Abcam, 1:4000, ab181602) as a loading control. Signal detection was performed using the ECL Kit (Beyotime Institute of Biotechnology) after incubation with an anti-rabbit or anti-mouse IgG secondary antibody (CoWin Bioscience Co., Beijing, China). Experiments were performed in triplicate.
4
Histopathological Analysis of Kidney Tissue
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Paraffin-embedded Sections (2 μm) of kidney were stained with periodic acid-Schiff (PAS), and Sirius red by routine protocol [19 (link)]. Immunohistochemistry was performed by routine method [20 (link)]. Primary antibodies include wnt3a (Abcam, ab28472), active-β-catenin (Millipore, 05-665), DKK1 (Abcam, 109416), AGT (Abcam, ab213705), ACE-1 (Abcam, ab222739), AT1 (Abcam, ab18801).
5
Western Blot Analysis of Signaling Proteins
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Western blotting was performed as described previously [46 (link)]. Aliquots of 15 μg cellular lysate protein were resolved by SDS-polyacrylamide gel electrophoresis (10% resolving gel) and subsequently transferred to a nitrocellulose membrane (AmerSham Biosciences, USA). Membranes were incubated with phospho-ERα (Ser122; Biorbyt, Cambridge, UK), phospho-ERα (Ser167; Abcam, UK), ERα (Abcam), phospho-GSK-3β (Ser9; Cell Signaling, Danvers, MA, USA), GSK-3β (Cell Signaling), active-β-catenin (Millipore, Molsheim, France), β-catenin (Millipore), and RUNX2 (Cell Signaling) antibodies overnight at 4 °C, respectively. To demonstrate equivalency of protein loading, a specific GAPDH antibody (Cell Signaling) was used. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-sheep or goat anti-mouse secondary antibody (Cell Signaling) for 1 h at room temperature. For the enhanced chemiluminescence reaction, immunoblots were developed in SuperSignal West Pico Chemilumemescent Substrate (Perbio Science, Pierce, Bonn, Germany), and the respective phosphoproteins or total proteins were visualized using the Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany).
6
Protein Expression Analysis in Gastric Cancer
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Total protein was extracted from gastric cancer cell lines and paired primary tumors and non-tumorous tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA). Twenty-microgram of protein mixed with 2 × SDS loading buffer were loaded on each lane, separated by 12% SDS-polyacrylamide gel electrophoresis. YY1 protein was then detected using anti-YY1 antibody (1:1000, H-10, sc-7341, Santa Cruz Biotechnology, Dallas, TX). Other antibodies applied included cleaved-PARP (Asp214) (1:1000, #9541, Cell Signaling, Danvers, MA), active-β-catenin (1:1000, #05-665, Millipore, Billerica, MA), β-catenin (1:10000, #610154, BD Transduction Laboratories, San Jose, CA), CCND1 (1:1000, #2926, Cell Signaling, Danvers, MA ), c-Myc (1:1000, #9402, Cell Signaling, Danvers, MA), anti-Mouse IgG-HRP (1:30000, #00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:40000, #00028856, Dako, Glostrup, Denmark).
7
Immunoblot Analysis of PD-L1 and Associated Proteins
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Cell protein extracts were prepared using M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions. Total protein was fractionated by SDS-PAGE and transferred onto PVDF membranes. The following primary antibodies were used: PD-L1 (17952-1-AP, ProteinTech), SOX6 (NBP1-85811, Novus), WNK2 (07-2261, Millipore), BTG3 (ab112938, Abcam), RBSP3 (ab106973, Abcam), active β-catenin (05-665, Millipore), OCT4 (ab19857, Abcam). p-AKT (sc-293125), p-ERK1/2 (sc-16982), total β-catenin (sc-7963), ERK1/2 (sc-514302), PTEN (sc-7974), cyclin D1 (sc-8396), p21 (sc-6246), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology. GAPDH was analyzed to show equal protein loading. Blots were developed with the enhanced chemiluminescence blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Immunoblot images were digitized and quantified using the ImageJ software. Results were expressed as a relative ratio of PD-L1 to GAPDH and the PD-L1/GAPDH ratio in normal cells was set as 1.
8
Immunofluorescence Analysis of Cell Adhesion and Cytoskeleton
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The samples on the nanofabricated coverslip were fixed with ice-cold 4% paraformaldehyde for 20 min, washed two times with phosphate-buffered saline (PBS), and permeablized with 0.1% Triton X-100 in PBS for 5 min. After washing with PBS, cultures were blocked with 10% goat serum for 1 h, and then incubated with primary antibodies against Vinculin (1:200, Sigma-Aldrich), E-cadherin (1:200, Cell Signaling, 3195), phospho-Myosin Light Chain (1:200, Cell Signaling, 3674), γ-tubulin (1:100, Abcam, ab11321), YAP (1:100, Cell Signaling, 4912 and Santa Cruz, sc-15407), active β-catenin (1:100, Millipore, 05-665), Vimentin (1:100, Abcam), Slug (1:100, Cell Signaling, 9585), Twist (1:100, Santa Cruz, sc-15393), Vimentin (1:100, Abcam, ab24525), and WT1 (1:100, Santa Cruz, sc-192) for 3 h at room temperature. After washing with secondary antibodies and Alexa Fluor 594 conjugated phalloidin (1:40, Molecular Probes) and Hoescht (Invitrogen), cultures were incubated for 1 h at room temperature. The slides were mounted with an anti-fade reagent (SlowFade gold, Invitrogen) and taken by an inverted microscope (Zeiss Axiovert 200 M) with an X40 oil immersion objective (Zeiss, 1.6 NA).
9
Comprehensive Antibody Characterization Protocol
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Anti-SSEA4, active β-catenin and total β-catenin monoclonal antibodies were purchased from Millipore (Billerica, MA, USA). Anti-Sca1-PE, CD34-PE, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3ε and CD28 antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE, CD178(FASL)-PE, Foxp3-PE, IL17-PE, and IFNγ-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-TERT, FASL and total β-catenin (ChIP grade) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-BRG1 antibody was purchased from Cell Signaling (Danvers, MA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
Signaling Pathways in Melanoma Cells
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Melanoma cells were lysed in RIPA buffer containing 50 mmol/l Tris-HCl pH 8.0, 150 mmol/l NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS supplemented with freshly added protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration was determined by Bradford assay. Cell lysates were diluted in 2x Laemmli buffer and protein samples (15 μg each) were loaded on 7% SDS-polyacrylamide gel. The proteins were transferred onto an Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated in a blocking solution: 5% nonfat milk in PBS-Tween 0.05% or 5% phosphoBLOCKER (Cell Biolabs, San Diego, CA, USA) in PBS-Tween 0.05%. Primary antibodies detecting PARP and total β-catenin were from Santa Cruz Biotechnology, active β-catenin (dephosphorylated on Ser37 and Thr41) from Millipore (Temecula, CA, USA), p-p65 (Ser536), p65, p-ERK1/2 (Thr202/Tyr204), ERK1/2 from Cell Signaling Technology (Danvers, MA, USA). Immunodetection of β-actin (Sigma-Aldrich) was used as a loading control.
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