For cytospin preparations, HT29 or Caco-2 colorectal cancer cells were harvested using Accutase (Sigma-Aldrich, Munich, Germany) and adjusted to a final concentration of 2 × 105 cells/ml. Cytospin preparations were performed with 50 μl of cell suspension at 550 rpm for one minute in a Cytospin4 cytocentrifuge (Thermo Fisher Scientific) and incubated with the following antibodies: PDGFRα (D1E1E) (Cell Signaling Technology) at a dilution of 1:500; PDGFRβ (28E1) (Cell Signaling Technology) at a dilution of 1:100; VEGFR1 (Y103) (Abcam, Cambridge, UK) at a dilution of 1:250; VEGFR2 (55B11) (Cell Signaling Technology) at a dilution of 1:200.
Vegfr2 55b11
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, Germany, United States, Czechia
VEGFR2 (55B11) is a laboratory reagent produced by Cell Signaling Technology. It is an antibody that specifically binds to the vascular endothelial growth factor receptor 2 (VEGFR2) protein. VEGFR2 is a receptor tyrosine kinase that plays a key role in angiogenesis and vascular development.
Automatically generated - may contain errors
Lab products found in correlation
Np 40 buffer, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Nonfat milk powder, by Merck Group (2 mentions)
Accutase, by Merck Group (1 mentions)
Cytospin 4 cytocentrifuge, by Thermo Fisher Scientific (1 mentions)
Pdgfrβ 28e1, by Cell Signaling Technology (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Phosstop, by Roche (1 mentions)
Chaps, by Merck Group (1 mentions)
Anti flag resin, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Prime western blotting detection reagent, by GE Healthcare (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Hif 1α antibody, by Cell Signaling Technology (1 mentions)
Vegfa, by Cell Signaling Technology (1 mentions)
Swine anti rabbit fitc f0205, by Agilent Technologies (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
7 protocols using vegfr2 55b11
1
Cytospin Preparation and Immunostaining of Colorectal Cancer Cells
2
Immunoprecipitation of Cadherins and VEGFR
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Confluent HUVECs in complete medium were rinsed with PBS containing 1 mM Ca2+ and 0.5 mM Mg2+, then lysed with cold 25 mM Tris, pH 7.5, 600 mM NaCl, 0.3% CHAPS (3023; Sigma-Aldrich), 0.15% Triton X-100, 1.5× PhosSTOP (Roche), and 1.5× Protease inhibitor (Roche) at 1 ml/106 cells. Lysates were drawn through a 23G syringe 15 times and incubated on ice for 30 min before clarification at 20,000 g for 10 min. Clarified lysates were immunoprecipitated with either anti-Flag resin (Sigma-Aldrich) or Protein A/G beads (Santa Cruz Biotechnology, Inc.) bound to anti-cadherin (VEcad, BV9 [Santa Cruz Biotechnology, Inc.]; Ncad, 610920 [BD]) or VEGFR (VEGFR2, 55B11 [Cell Signaling Technologies]; VEGFR3, AF349 [R&D Systems]) antibodies, as indicated, for 2.5 h at 4°C. Beads were washed three times with 1 ml of lysis buffer. Flag immunoprecipitates were eluted at 4°C for 60 min in lysis buffer with 0.2 mg/ml 3×-flag peptide (Sigma-Aldrich). Eluted proteins were then collected in protein sample buffer (PSB), then analyzed by SDS-PAGE and immunoblotting with chemiluminescent HRP detection.
3
Western Blot Analysis of VEGFR2 and ASM
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Cells were lysed with NP-40 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,400 rpm at 4 °C and the supernatant was collected. Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to nitrocellulose membranes (GE Healthcare Life Science). Non-specific binding was blocked with 5% non-fat milk powder (Sigma-Aldrich) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated with rabbit anti-vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) (55B11; Cell Signaling Technology), goat anti-ASM (AF5348, R&D Systems) and rabbit anti-β-actin (4967; Cell Signaling Technology) antibody overnight at 4 °C, rinsed and incubated in peroxidase-conjugated secondary antibodies (Santa Cruz, Heidelberg, Germany) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science). VEGFR2 and ASM expression were normalized to β-actin abundance.
4
VEGF-A Regulation of EZH2 and HIF-1α
5
Immunofluorescence Staining of HUVECs and rMCs
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HUVECs and rMCs were cultured according to experimental conditions and subsequently washed with PBS and fixed with 4% Histofix for 10 min, after which they were incubated with a permeabilization/blocking solution (2.5% BSA and 0.3% Triton-X in PBS) for 1 h at room temperature. The cells were further incubated with 200 μL of primary antibody solution (VEGFR2 55B11, Cell Signaling; Ang2 SC74403, Santa Cruz; GFAP Z0334, Dako; diluted 1:200 in PBS) overnight at 4 °C, then washed thrice with PBS and incubated with the secondary antibodies (swine anti-rabbit FITC, F0205, Dako; diluted 1:20 in PBS) for 1 h at room temperature in the dark. The cells were subsequently washed thrice with PBS, incubated with DAPI (1 μg/mL) for 10 min, and washed twice with PBS. They then were covered with Roti FluorCare mounting medium and covered with coverslips. The images were obtained using a Leica SP8 confocal microscope, and fluorescence intensity quantification was done using ImageJ.
6
Antibody Panel for Angiogenesis Signaling
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Primary antibodies against VE-cadherin (#2158), phospho-VEGFR2 at Tyr1175 (D5B11, #3770), VEGFR2 (55B11, #2479), phospho-AKT at Ser473 (D9E, #4060), AKT (C67E7, #4691), phospho-ERK1/2 (p44/42 MAPK, 137F5, #4695), GAPDH (D16H11, #5174) and pan-Actin (D18C11, #8456) were from Cell Signalling Technology (CST; Danvers, MA, USA). The antibody anti-RCAN1 (#D6694) was from Sigma-Aldrich (St. Louis, MO, USA), anti-CD81 (MCA1847) was from BioRad (Hercules, CA, USA), anti-CD9 (#10626D) was from ThermoFisher (Waltham, MA, USA), anti-CD63 (#556019) was from BDBiosciences (Franklin Lakes, NJ, USA), and anti-GRP78 was from Serotec (Raleigh, NC, USA). Secondary anti-rabbit or anti-mouse Horseradish Peroxidase (HRP)-coupled antibodies were from Jackson Immunoresearch (West Grove, PA, USA) and anti-rabbit Alexa488 antibody (#A-21206) was from Invitrogen (Waltham, MA, USA).
Human recombinant VEGFA165 was obtained from Proteintech (#HZ-1038; Rosemont, IL, USA).
Human recombinant VEGFA165 was obtained from Proteintech (#HZ-1038; Rosemont, IL, USA).
7
Shedding Light on Angiogenic Factors
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Cells were lysed with NP-40 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,400 rpm at 4°C and the supernatant was collected. Equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose membranes (GE Healthcare Life Science). Non-specific binding was blocked with 5% non-fat milk powder (Sigma-Aldrich) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated with rabbit anti-vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) (55B11; Cell Signaling Technology), goat anti-ASM (AF5348, R&D Systems) and rabbit anti-β-actin (4967; Acid sphingomyelinase deactivation post-ischemia/ reperfusion promotes angiogenesis/ 26 Amnis ImagestreamX flow cytometry of sEVs sEVs were quantified with an ImageStreamX MkII instrument (Merck Millipore) as described previously 55 after CD9-FITC (MEM-61; Exbio, Vestec, Czech Republic) and CD63-APC (MEM-259; Exbio) antibody staining. All samples were appropriately diluted in order to avoid coincidence or swarm detection. Data analysis was performed using Amnis IDEAS software (version 6.1).
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