WB experiments were performed as previously reported [69 (link),93 (link)] using the following antibodies: anti-β-Actin antibody (4967; Cell Signaling Technology, Danvers, Massachusetts), anti-NICD antibody (ab83232; Abcam, Cambridge, UK), anti-Myc antibody (06–549; Millipore), anti-Flag antibody (F7425; Sigma-Aldrich), anti-DLL4 antibody (ab7280; Abcam), anti-JAG1 antibody (ab7771; Abcam), anti-Notch1 antibody (3608; Cell Signaling Technology), anti-Erk antibody (9102; Cell Signaling Technology), anti-p-Erk antibody (9101; Cell Signaling Technology), anti-p-AKT antibody (4060; Cell Signaling Technology), and anti-AKT antibody (9272; Cell Signaling Technology). Quantification of protein level using gray analysis (Gel-Pro analyzer). Immunofluorescence experiments were performed as previously reported [94 (link)] using the following antibodies: anti-Notch1 antibody (3608; Cell Signaling Technology), anti-Notch1 antibody (3447; Cell Signaling Technology), anti-DLL4 antibody (ab7280; Abcam), anti-JAG1 antibody (ab7771; Abcam), anti-LAMP1 antibody (15665; Cell Signaling Technology), anti-EEA1 antibody (610457; BD Biosciences, Franklin Lakes, New Jersey), anti-APPL1 antibody (3858; Cell Signaling Technology), anti-AKT antibody (2920; Cell Signaling Technology), anti-AKT antibody (9272; Cell Signaling Technology), and anti-PIK3CA antibody (ab40776; Abcam).
Ab7280
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab7280 is an anti-mouse CD4 antibody that can be used for flow cytometry applications. It recognizes the CD4 surface antigen expressed on T helper cells.
Automatically generated - may contain errors
Lab products found in correlation
Ab7771, by Abcam (2 mentions)
Sc 373891, by Santa Cruz Biotechnology (2 mentions)
Sc 5545, by Santa Cruz Biotechnology (2 mentions)
C651 56 dbhn c, by Developmental Studies Hybridoma Bank (2 mentions)
Ab52627, by Abcam (2 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti myc antibody, by Merck Group (1 mentions)
Anti erk antibody, by Cell Signaling Technology (1 mentions)
Anti p akt antibody, by Cell Signaling Technology (1 mentions)
Anti akt antibody, by Cell Signaling Technology (1 mentions)
Ab83232, by Abcam (1 mentions)
Ab40776, by Abcam (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Anti lamp1 antibody, by Cell Signaling Technology (1 mentions)
Anti p erk antibody, by Cell Signaling Technology (1 mentions)
Anti eea1 antibody, by BD (1 mentions)
Anti nicd antibody, by Abcam (1 mentions)
Anti notch1 antibody, by Cell Signaling Technology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
15 protocols using ab7280
1
Western Blot and Immunofluorescence Protocols
2
Western Blot Analysis of Notch Pathway
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Cells were washed with ice-cold PBS and lysed in RIPA buffer [50 mmol/l Tris (pH 7.5), 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)] containing phenylmethylsulfonyl fluoride (PMSF; 1 mmol/l) and protease inhibitors (2 g/ml; Protease inhibitor cocktail set III, Calbiochem, Billerica, MA, USA) on ice for 30 min. The lysates were clarified by centrifugation at 13,000 × g for 30 min at 4°C. The total protein concentration was estimated using a Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel, transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), blocked and probed with antibodies against β-catenin (1:1,000; sc-65480; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), c-myc (1:1,000; sc-40; Santa Cruz Biotechnology, Inc.), Jagged (1:1,000; sc-390177; Santa Cruz Biotechnology, Inc.), Notch1 (1:1,000; sc-373891; Santa Cruz Biotechnology, Inc.), Notch2 (1:1,000; sc-5545; Santa Cruz Biotechnology, Inc.), DLL4 (1:1,000; ab7280; Abcam, Cambridge, MA, USA) and Pra-1 (1:1,000; ab76413; Abcam). Upon washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by super enhanced chemiluminescence detection reagent (Applygen, Beijing, China).
3
Protein Expression Analysis for Angiogenesis
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SPECs and FOS were collected after 1, 2, and 3 days of culture in 2% linear agarose molds as previously described. Samples were snap frozen and mechanically homogenized in RIPA lysis buffer with protease inhibitor cocktail. Samples were maintained in constant agitation for 2 h at 4°C and centrifugated for 20 min at 16,000 g at 4°C. Supernatant was stored in fresh tube at −20°C. Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific 23227) was used to estimate protein concentration for samples as per manufacturer's instructions. Before gel electrophoresis, samples were diluted in RIPA buffer to attain 20 μg of protein in 20 μL solution, and further diluted 1:1 in 2 × Laemmli Sample buffer to attain 40 μL loading volumes. Samples were loaded onto Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels. Following protein separation and overnight transfer onto PVDF membranes, western blots were performed using antibodies toward GAPDH (loading control) (Calbiochem CB1001; 1:1000), VEGFR2 (Abcam; ab39256, 1:900), VE-Cadherin (ThermoFisher Scientific 36-1900; 1:250), vWF (Abcam; ab6994, 1:500), and DLL4 (Abcam; ab7280, 1:1000).
4
Multiparametric Flow Cytometry of Vascular and Lymphatic Endothelial Cells
5
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemical staining of FFPE tumour tissue was performing using rabbit polyclonal antibodies against DLL4 (ab7280, Abcam, UK; dilution 1 : 200), Notch1 (ab27526, Abcam, UK; dilution 1 : 100), Notch3 (sc5593, SantaCruz, US; dilution 1 : 25) and Vascular Endothelial Growth Factor (VEGF) (rb9031, Labvision corporation, Belgium; dilution1 : 100). Antigen retrieval was performed by microwave pre-treatment in EDTA buffer for 15 min for Notch3 and in citrate buffer for 10 min for DLL4, Notch1 and VEGF. Renal tissue was used as positive control for DLL4 expression, colon tissue for Notch1 and Notch3, and colon cancer tissue for VEGF expression. Irrelevant rabbit IgG antibodies were applied to negative control.
6
Immunofluorescence Imaging of Notch Signaling
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For immunofluorescence imaging, tissue sections were deparaffinized and antigen retrieval was performed with boiling citrate buffer (10 mM in PBS, pH 6) for detection of Notch1 and Dll4. Primary antibodies used were anti-Notch1 (D1E11) rabbit monoclonal (1:200; Cell Signaling #3608), anti-Notch2 (1:100; C651-56 DbHN-C, Developmental Studies Hybridoma Bank), anti-Dll1 (1:100; Abcam #ab84620), and anti-Dll4 rabbit polyclonal (1:100; Abcam #ab7280) with fluorescently tagged secondary antibodies purchased from Invitrogen, applied at 1:400 dilutions. Images were captured with 20X or 40X objective on a Zeiss LSM880 confocal microscope with ZEN software (Carl Zeiss Microscopy) for acquisition. Image processing was performed using Imaris software (Bitplane).
7
Shear Stress-Induced Protein Expression
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Cells grown in glass slides were treated with shear stress for 2 h. Then total protein was obtained by using M-PER protein extraction buffer, and quantified using a BCA kit (Thermo Fisher Scientific, Inc.) and separated on 7.5 or 12% polyacrylamide gel followed by transfer onto an ImmunBlot PVDF membrane (GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were blocked for 1 h with 5% BSA in Tris-phosphate buffer containing 0.05% Tween-20 (TBS-T). It was further incubated overnight at 4°C with anti-Notch1 (1:2,000; ab52627), anti-DLL4 (1:1,000; ab7280), anti-Hey1 (2 μg/ml, ab22614), anti-Hey2 (2 μg/ml; ab25404) all from Abcam (MA, USA); anti-EphrinB2 (1:1,000; sc-398735) and anti-EphB4 (1:1,000; sc-130081) both from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-VEGFR2 (1:2,000; ab39256) and anti-CD31 (1:500; ab28364) both from Abcam, primary antibodies. After three washes (5 min) with TBS-T, membranes were further incubated with HRP-conjugated secondary antibodies: anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) both from Cell Signaling Technology, Inc., (Danvers, MA, USA) for 1-2 h and followed by three washes with TBS-T. The target protein signal was detected and digitized using ECL (Thermo Fisher Scientific, Inc.).
8
Multiparametric Immunostaining of Lung Tissue
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Antibodies for staining: mouse anti-DLL4 (sc-365429, SCBT) and anti-PECAM (sc-376764, SCBT); rabbit anti-ERG (ab92513, Abcam), anti-AQP5 (orb15125, Biorbyt), anti-APLNR (20341-1-AP, Thermo Fisher Scientific), anti-SPC (ab90716, Abcam), anti-PDGFRA (ab203491, Abcam), anti-JAG1 (ab7771, Abcam); rat anti-PECAM (550274, BD Biosciences), anti-Ki67 (11-5698-82, Thermo Fisher Scientific); goat anti-Carbonic Anhydrase IV (CAR4) (PA5-47312, Thermo Fisher Scientific); and chicken anti-β galactosidase (ab9361, Abcam). Antibodies for Western blot: mouse anti-DLL4 (sc-365429, SCBT), anti-HES1 (sc-166410, SCBT), anti-JAG1 (sc-390177, SCBT), anti-PECAM (sc-376764, SCBT), anti-NR2F2 (sc-271940, SCBT); rabbit anti-NICD (4147, Cell Signaling), anti-PDGFRA (ab203491, Abcam), anti-SPC (ab90716, Abcam), and anti-VEGFA (ab46154, Abcam); goat anti-DLL4 (ab7280, Abcam); anti-mouse β-actin (ACTB) (A1978, Sigma). The primers were purchased commercially from Sigma.
9
Immunohistochemical Analysis of DLL4 Protein
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Tumor specimens were fixed in 10% of formaldehyde and embedded in paraffin for histological sections. Paraffin-embedded sections were deparaffinized in xylene, dehydrated in graded alcohol. Immunohistochemistry for DLL4 protein was performed using a rabbit monoclonal antibody (Abcam, ab7280, Cambridge, UK). Antigen retrieval was performed in citrate buffer pH 6.0, then the sections were incubated overnight at 4°C with the primary antibody at 1:200. Next, they were rinsed with phosphate buffer solution (PBS) and incubated with the horseradish peroxidase-conjugated secondary antibody, followed by a rinse in PBS, incubation with diaminobenzidine staining, and counterstaining with hematoxylin blue. The negative control sections were incubated with PBS in equal concentrations to the primary antibody, and known positive human kidney tissue was performed as positive control.
10
Protein Expression Analysis of Notch Pathway
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The cells at passage 3 were rinsed twice with precooled PBS, then 1 mL of cell lysis buffer (50.0 mmol/L Tris pH = 7.6, 150.0 mmol/L NaCl, 0.1% SDS, 1.0% NP-40, protease inhibitor cocktail) was added, and the cells were scraped off. The cells were lysed at 4°C for 30 min under rotation and centrifuged at 15000 rpm for 30 min, and the supernatant was collected. Protein concentrations were determined by the BCA Protein Assay Reagent (Thermo Fisher Scientific, Rockford, IL, USA), after which 25 μg of total proteins was loaded to 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (PVDF, Millipore) using the conventional method. The membrane was immunoblotted with primary antibodies against CD146 (1 : 500, ab75769, Abcam), Jagged1 (1 : 500, ab109536, Abcam), DLL4 (1 : 500, ab7280, Abcam), or GAPDH (1 : 10000, AP0063, Bioworld technology), followed by incubation with a goat anti-mouse (1 : 10000, BS12478, Bioworld technology) or goat anti-rabbit (1 : 10000, A0545, Sigma) secondary antibody. The bands were detected using an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis of each band was performed with Quantity-one (Bio-Rad) software.
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