All the embedded PCa and CRPC samples were cut into 5-µm-thick sections. The immunoreactivities of SOX17, Notch1, Notch2, Notch3, Notch4 were detected by an immunoperoxidase staining procedure with the primary antibodies (anti-SOX17, 1:200, Abcam, cat.no.ab192453; anti-Notch1, 1:200, Abcam, cat. no.ab8925; anti-Notch2, 1:200, Cell Signaling Technology, cat.no. D76A6; anti-Notch3, 1:200, Abcam, cat.no. ab23426; anti-Notch4, 1:200, Santa Cruz Biotechnology, cat.no. sc-377399 ). Staining scoring, according to staining intensity, was defined as 0, no staining; 1, weak staining; 2, light staining; 3, moderate staining; and 4, strong staining. Staining scores of ≤1 were defined as negative expression, while staining scores of ≥2 were defined as positive expression.
Anti notch2
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Anti-Notch2 is a primary antibody that specifically binds to the Notch2 protein, a cell surface receptor involved in cell-cell communication and signaling pathways. It can be used for the detection and analysis of the Notch2 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti notch3, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti notch1, by Cell Signaling Technology (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti notch3, by Abcam (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti notch4, by Santa Cruz Biotechnology (1 mentions)
Anti sox17, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
Tcs spe confocal microscope, by Leica (1 mentions)
Anti mapk1, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti cd133, by GeneTex (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti notch2
1
Immunoreactivity of Notch Signaling Pathway
2
Immunofluorescence Staining and Imaging
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Cultures were grown on chamber slides and fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS then stained using standard protocols. Briefly, the fixed cells were permeabilized with 0.1% Triton X-100 then blocked using 10% serum. The primary antibodies used were anti-Mapk1 (1:800), anti-phospho-Neu (1:50)(Santa Cruz Biotech), anti-Notch2 (1:200)(Cell Signaling Technologies); each was applied and incubated overnight at 4°C. Secondary antibodies were conjugated to Alexa 488 or Alexa 568 (1:200)(Life Technologies) and incubated for 60 min at room temperature. Slides were coverslipped with ProLong Antidfade mounting media containing DAPI (Life Technologies). Images were collected using a Leica TCS SPE confocal microscope at the same laser and acquisition setting. To ensure the laser power and imaging settings were not acquiring autofluoresence, a comparison image was generated from using an unstained sample. Images were analyzed using Image J and Adobe Photoshop.
3
Protein Expression Analysis of A549 Cells
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For Western blot analysis, samples of the A549 cells induced by the small molecule chemical compounds were harvested, lysed in 200 μL RIPA lysis buffer (P0013B Beyotime, Shanghai, China), and centrifuged at 12000 rpm for 3 min. The cell lysate protein was quantified using the BCA Protein Assay Kit (Thermo Fisher). Then 15 or 20 μg of total protein sample were resolved on a 10% acrylamide gel for SDS-polyacrylamide gel electrophoresis and electrotransferred onto a PVDF membrane (Millipore, Darmstadt, Germany) at 150 mA for 120 min (Bio-Rad). Anti-CD133 (1:1000, GeneTex, Irvine, CA, USA), anti-vimentin (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (1:1000, Cell Signaling Technology), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-NOTCH1 (1:1000, Cell Signaling Technology), anti-NOTCH2 (1:1000, Cell Signaling Technology), anti-NOTCH3 (1:1000, Cell Signaling Technology), anti-HES1 (1:1000, Abcam, Cambridge, UK), GAPDH antibody (1:10000, Abcam), and horse radish peroxidase-conjugated goat-anti-rabbit IgG (1:1000, Cell Signaling Technology) antibodies were used. Pierce ECL liquid (Thermo Fisher) was used for exposure, which was performed using a gel imaging system (Bio-Rad).
4
Protein Extraction and Western Blot Analysis
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Total protein was obtained by RIPA lysis buffer (Beyotime) with protease inhibitor cocktail (Thermo Fisher), and protein concentrations were measured by Pierce BCA Protein Assay Kit (Thermo Fisher). A total of 30‐40 ng protein extracts were loaded onto SDS‐PAGE gels (6%‐12%) and then transferred to PVDF membranes. PVDF membranes were blocked by 5% w/v non‐fat dry milk which was dissolved in tris‐buffered saline solution containing 0.1% tween‐20 (TBS‐T) for 2 hours. The PVDF membranes were then incubated with the primary antibodies at 4°C overnight: anti‐NOTCH2 (1:4000 dilution; Cell Signaling Technology), anti‐phospho‐Histone H3 (PHH3) (1:2000 dilution; Cell Signaling Technology), anti‐PCNA (1:2000 dilution; Cell Signaling Technology) and anti‐β‐actin (1:4000 dilution; Cell Signaling Technology). After three washing with TBS‐T, the PVDF membranes were incubated with HRP‐labelled goat anti‐rabbit IgG secondary antibody (1:8000 dilution; Abcam) for 1 hour at room temperature. After the final washing with TBS‐T, protein bands were visualized with ECL (Merck Millipore) reagents.
5
Immunofluorescence Staining of Notch Receptors
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MGECs or MMECs (5 × 103) were seeded on chamber slides (Lab Tek II Chamber Slides, Thermo Scientific Fisher Scientific Inc.), fixed, permeabilized, and incubated overnight with anti-Notch1 and anti-Notch2 (Cell Signaling Technology Inc., Danvers, MA) primary antibodies. The next day, ECs were incubated with secondary FITC-conjugated anti-rabbit antibody (Sigma-Aldrich). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen Corp.).
6
Quantitative Western Blot Analysis of Cell Signaling Proteins
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For western blot analysis, cell lysates were prepared from cell lines with RIPA lysis buffer kit (Santa Cruz Biotechnology, Santa Cruz, CA), and the protein concentrations were quantified using a Bio-Rad protein assay (Bio-Rad, Hercules, CA). Whole-cell proteins (30 µg) were separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Amersham Corp., Arlington Heights, IL). The membranes were incubated with primary antibodies anti-IRF4 (1:500, cat. sc-6059; Santa Cruz Biotechnology), anti-Notch1 (1:100, cat. Val1744, Cell Signaling, Danvers, MA), anti-Notch2 (1:500, cat. D67C8; Cell Signaling), anti-AKT (1:500, cat. 9272; Cell Signaling), anti-AKT phosphorylation (1:500, cat. 9271; Cell Signaling) or anti-GAPDH (1:1,000, cat. 2111; Cell Signaling) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibodies (1:1,000, cat. A50-106P; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China;) were subsequently used at room temperature for 1 h. Signals were detected using enhanced chemiluminescence kit (Wuhan Booute Biotechnology Co., Ltd, Wuhan, China; cat. no. orb90504) and exposed to Kodak X-OMAT film (Eastman Kodak, Rochester, NY, USA).
7
Protein Extraction and Western Blot
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Protein lysates were collected using M-PER mammalian protein extraction reagent with protease and phosphatase inhibitors added (Thermo Scientific; Rockford, IL). Lysates were cleared at 10,000 rpm for 10 min at 4°C then transferred to new tubes and frozen at -20°C until analysis. Protein concentration was determined using a BCA assay. Samples were combined with sample running buffer and boiled for 10 min. Proteins were separated on a 4–20% gel, transferred to nitrocellulose then probed using anti-β -actin and anti-Notch2 followed by HRP-conjugated secondary antibodies (all Cell Signaling Tech). Target proteins were detected using the chemiluminescent reagent Lumiglo (Cell Signaling) and visualized using FluorChemM (Cell Biosciences; Wallingford, CT).
8
Western Blot Analysis of Notch Signaling
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Western blotting was performed as previously described (Zhao et al., 2014 (link)). The blots were probed with the following primary antibodies: anti-Notch2 (Cell Signaling Technologies) at 1:1000 dilution, anti-Notch3 (Santa Cruz Biotechnology) at 1:200, anti-NF-κB p65 (Cell Signaling Technologies) at 1:1000, anti-cyclin D1 (Cell Signaling Technologies) at 1:1000, anti-c-Myc (Abcam,) at 1:5000, anti- MMP-2 (Abcam) at 1:1000, anti-GAPDH (Affinity Biosciences, Cincinnati, OH, USA) at 1:5000, and anti-β-actin (KangChen, Shanghai, China) at 1:5000. Blots were detected using the Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA) and visualized by FluorChem®HD2 (ProteinSimple, San Jose, CA, USA).
9
Comprehensive Nuclear Protein Analysis
10
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from WM-266-4 cells treated with the IC50 of ORT, DTIC, or the ORT/DTIC combination, using the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Inc.). Protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). For each group, 25 µg of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 10–12% gel. Next, the resolved proteins were transferred onto polyvinylidene fluoride membranes (0.45 µm; EMD Millipore). The membranes were blocked with 5% skim milk or 5% bovine serum albumin and incubated overnight at 4°C with anti-β-actin, anti-BAX, anti-CASP3, anti-CASP9, anti-MYC, anti-CCND1, anti-NOTCH2 and anti-NOTCH3 antibodies diluted 1,000-fold (Cell Signaling Technology). Then, the membranes were probed with secondary antibodies, diluted 1,000-fold, for 2 h at room temperature. The antigen-antibody complexes were visualized using enhanced chemiluminescence western blotting detection reagents (Thermo Fisher Scientific, Inc.). ChemiDoc XRS (Bio-Rad) and the Image Lab software (version 4.1; Bio-Rad) were used to analyze the blot images.
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