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Total vegfr2

Manufactured by Cell Signaling Technology
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Total VEGFR2 is a laboratory reagent that measures the total amount of VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) protein in cell lysates or tissue samples. VEGFR2 is a key receptor involved in angiogenesis and vascular permeability. The Total VEGFR2 product provides a quantitative assessment of VEGFR2 expression levels.

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Lab products found in correlation

Gapdh, by Abcam (2 mentions) Total akt, by Cell Signaling Technology (2 mentions) Pvegfr2 y1059, by Cell Signaling Technology (2 mentions) Pvegfr2 y1059, by Merck Group (2 mentions) Pvegfr2 y951, by Cell Signaling Technology (2 mentions) Ve cad, by Santa Cruz Biotechnology (2 mentions) Sc 9989, by BD (2 mentions) Ve cad, by BD (2 mentions) Sc 1718 r, by BD (2 mentions) Goat anti vegfr2, by R&D Systems (2 mentions) Rabbit anti ve cadherin, by Cell Signaling Technology (2 mentions) Pvegfr2 y1175, by Cell Signaling Technology (2 mentions) Rabbit anti rab5, by Cell Signaling Technology (2 mentions) Rabbit anti ha, by Cell Signaling Technology (2 mentions) Ha tag, by Cell Signaling Technology (2 mentions) Mouse anti ha 11, by Fortrea (2 mentions) Lysis buffer, by Cell Signaling Technology (1 mentions) P pdgfrβ, by Cell Signaling Technology (1 mentions) P src tyr416, by Cell Signaling Technology (1 mentions) Pdgf bb, by Merck Group (1 mentions)

Close spelling variants of this product

VEGFR2 (164 citations)

8 protocols using total vegfr2

1

Protein Expression and Immunoblotting

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Cells were lysed with lysis buffer (catalog no. #9803s; Cell Signaling Technology) containing protease inhibitor cocktail (catalog no. #3100-001; Gendepot). Total protein was immunoblotted with primary antibodies against KAI1 (catalog no. ab135779; Abcam), LIF (catalog no. AB-449-NA; R&D Systems), tSrc, pSrc (Tyr416), and pSrc (Tyr527) (Cell Signaling Technology), Myc (catalog no. 05-724; Sigma-Aldrich), p53 (catalog no. MABE327; Merck Millipore), Pbx1 (catalog no. sc-889; Santa Cruz), Cav-1 (Caveolin-1, catalog no. sc-894; Santa Cruz), Flot1 (Flotillin1, catalog no. #3253s; Cell signaling Technology), p-VEGFR2 (catalog no. ab38473; Abcam), total VEGFR2 (catalog no. #2479s; Cell Signaling Technology), p-PDGFRβ (catalog no. #3166s; Cell signaling Technology), total PDGFRβ (catalog no. #4564s; Cell signaling Technology), PDGF-BB (catolg no. 07-1437; Sigma-Aldrich), and VEGF-A (catalog no. #ABS82; Merck Millipore) followed by incubation in horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratory). GAPDH (catalog no. ab9485; Abcam) and beta-actin (catalog no. ab8227; Abcam) were used as an internal control.
Lee J.W., Hur J., Kwon Y.W., Chae C.W., Choi J.I., Hwang I., Yun J.Y., Kang J.A., Choi Y.E., Kim Y.H., Lee S.E., Lee C., Jo D.H., Seok H., Cho B.S., Baek S.H, & Kim H.S. (2021). KAI1(CD82) is a key molecule to control angiogenesis and switch angiogenic milieu to quiescent state. Journal of Hematology & Oncology, 14, 148.
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2

Western Blot Analysis of VEGFR2, ERK1/2, and HIF-1α

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Tumor tissues were lysed, and the protein content was measured by Western blotting. The membranes were incubated with primary antibodies against total VEGFR2 (1:1000; Cell Signaling, Danvers, MA, USA), total and phosphor-ERK1/2 (1:1000; Cell Signaling), HIF-1α (1:1000; Cell Signaling Technologies, USA) and GAPDH (1:5000; Cell Signaling Technologies, USA) followed by goat anti-rabbit (1:20,000) antibodies conjugated with horseradish peroxidase (Cell Signaling Technologies, USA). Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) was employed to detect the signals using a FluorChem M Simple Imager (Protein Simple, USA).
Kanugula A.K., Adapala R.K., Jamaiyar A., Lenkey N., Guarino B.D., Liedtke W., Yin L., Paruchuri S, & Thodeti C.K. (2021). Endothelial TRPV4 channels prevent tumor growth and metastasis via modulation of tumor angiogenesis and vascular integrity. Angiogenesis, 24(3), 647-656.
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3

Western Blot Analysis of Signaling Pathways

Cited 1 time
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Unstimulated or stimulated endothelial cells, and serum (10%) stimulated tumor cells were treated with 2-DG or other agents, incubated for different time periods and lysed. Protein concentration was determined from lysates by the BCA assay (Thermo Scientific, Rockford IL). 10-20 μg proteins were prepared with 4X Laemmeli sample buffer and separated in 10% or 4-20% Mini-Protean TGX gel (BioRad, Richmond, CA), transferred to 0.45 μm pore sized PVDF membranes (Bio-Rad) and probed (1:1000 dilution) for Akt pSer473, S6 pSer240/244, Erk pThr202/Tyr204, GSK-3β pSer9, PERK pThr980, cleaved-Caspase-3, cleaved-PARP, total-VEGFR2, VEGFR2 pTyr1175, and PLC-γ1 pTyr783 (Cell signaling, Danvers, MA), GSK-3β pTyr216 (Calbiochem), and GAPDH (1:20,000 dilution) for loading control (Rockland, Gilbertsville, PA). Following probing, membranes were processed as previously described (15 (link), 18 (link)). Band intensities of replicate Western blot figures were quantified with ImageJ software, normalized to the corresponding GAPDH bands, and results presented as percentage of control (untreated cells) +/− SEM.
Kovács K., Decatur C., Toro M., Pham D.G., Liu H., Jing Y., Murray T.G., Lampidis T.J, & Merchan J.R. (2015). 2-DEOXY-GLUCOSE DOWN REGULATES ENDOTHELIAL AKT AND ERK VIA INTERFERENCE WITH N-LINKED GLYCOSYLATION, INDUCTION OF ENDOPLASMIC RETICULUM STRESS AND GSK-3β ACTIVATION. Molecular cancer therapeutics, 15(2), 264-275.
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4

VEGFR2 Activation by VEGF-A and HB-002.1

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4 ml of human umbilical vein endothelial cells (HUVECs) (Cat#HUVEC-004, ALLCELLS) in complete HUVEC-adapted medium (Cat#H-004, ALLCELLS) were incubated in 6 cm dishes at 37°C, 5% CO2 for 24 hours, cells were starved for 2 hours and then challenged for 15 minutes with either medium alone, or VEGF-A (20 ng/ml) only, or VEGF-A pre-incubated with varying amounts of HB-002.1. Cells were washed twice with cold PBS and then dissolved in 200 μl of lysis buffer (50 mM Tris, pH 7.4, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, pH 8.0, 150 mM NaCl). After centrifugation and quantitation, equal amounts of supernatant from each sample were subjected to Western blotting analysis using antibodies specific either for total VEGFR2 (Cat# 2479, Cell Signaling Technology) or for VEGFR2 phosphotyrosine (Cat# 3770S, Cell Signaling Technology).
Liu L., Yu H., Huang X., Tan H., Li S., Luo Y., Zhang L., Jiang S., Jia H., Xiong Y., Zhang R., Huang Y., Chu C.C, & Tian W. (2015). A novel engineered VEGF blocker with an excellent pharmacokinetic profile and robust anti-tumor activity. BMC Cancer, 15, 170.
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5

Structural Modification and Evaluation of Asiatic Acid Derivatives

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AA was extracted from C. asiatica (L.) Urban and structurally modified in our laboratory to produce AA-PMe.20 (link) AA and AA-PMe were both dissolved in 1% dimethyl sulfoxide (DMSO; Sigma-Aldrich Co, St Louis, MO, USA) and their structures are presented in Figure 1. Endogenous alkaline phosphatase (EAP) staining was tested by a phosphatase substrate kit (Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Recombinant human VEGF was purchased from Thermo Fisher Scientific. Primary antibodies for total Akt, pAkt-ser473, total ERK1/2, p-pERK1/2, total VEGFR2, and pVEGFR2-Tyr1175 were brought from Cell Signaling Technology (Danvers, MA, USA). GAPDH and tubulin antibody were from Abcam (Cambridge, UK).
Jing Y., Wang G., Xiao Q., Zhou Y., Wei Y, & Gong Z. (2018). Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo. OncoTargets and therapy, 11, 1871-1884.
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6

Antibody Panel for Angiogenic Signaling

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The following antibodies were from Cell Signalling Technology (Frankfurt, Germany): Total VEGFR2 (#2479), phospho(Y1175)-VEGFR2 (#2478), total VEGFR1 (#64,094), phospho(S1177)-eNOS (#9570), total Akt (#4691), phospho(S473)-Akt (#9271), total Erk (#9107), phospho(T202/Y204)-Erk (#9106), total AMP-activated protein kinase α (AMPKα) (#2532), phospho(T172)-AMPKα (#2531), total p38 (#9212), phospho(T180/Y182)-p38 (#4511), phospho(Y207)-Crk-L (#3181), phospho(Y1086)-EGFR (#2220), total EGFR (#2232). Total eNOS antibody (#610,297) and total ABL-1 antibody were purchased from BD Biosciences (Heidelberg, Germany). Total ABL-2 (#sc-81,154) and total Crk-L (#sc-365,092) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, US). Total FRK antibody (#A95,485) was from Antibodies.com (Stockholm, Sweden). Horseradish peroxidase (HRP)-conjugated secondary antibodies to rabbit or mouse IgG were from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD, USA).
Zibrova D., Ernst T., Hochhaus A, & Heller R. (2024). The BCR::ABL1 tyrosine kinase inhibitors ponatinib and nilotinib differentially affect endothelial angiogenesis and signalling. Molecular and cellular biochemistry.
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7

Antibody Panel for Vascular Signaling Analysis

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List of antibodies with application and dilutions (IHC, immunohistochemistry; WB; western blot): pVEGFR2 Y1175 (WB 1:1000, Cell Signaling #2478), pVEGFR2 Y1059 (for HUVEC, WB 1:1000, Cell Signaling #3817, ), pVEGFR2 Y1059 (for mouse ECs, WB 1:1000, EMD Millipore #ABS553), pVEGFR2 Y951 (WB 1:1000, Cell Signaling #4991 ), VEGFR2 total (WB 1:1000, Cell Signaling #2479), VE-Cad (for HUVEC, WB 1:200, Santa Cruz #sc-9989 (F-8) ), VE-Cad (for mouse ECs, WB 1:500, BD Pharmingen #555289), mouse Sdc2 (WB 1:200, R&D #AF6585 - polyclonal raised against mouse Sdc2 ED), human Sdc2 (WB 1:200, R&D # AF2965, polyclonal raised against mouse human ED), mouse PTP1b (WB 1:200, Santa Cruz #sc-1718-R (N19-R)), human PTP1b (WB 1:500, BD Pharmingen #610139), VEPTP ( VE-PTP-C pAb69 and VE-PTP 1–8 pAb70 were gifts from Prof. Dietmar Vestweber, Max Plank Institute for Molecular Biomedicine, Münster, Germany), DEP1 (WB 1:200, Santa Cruz #sc-21761 (143–41)) NRP1 (WB 1:1000, Cell signaling #3725,), HA-tag (WB 1:1000, Cell signaling #3724), rabbit anti-Rab5 (Cell Signaling, #3547, IHC 1:200); goat anti-VEGFR2 (R&D, #AF357, IHC 1:100); mouse anti-HA.11 (Covance, #MMS-101P, IHC 1:200); rabbit anti-HA (Cell signaling, #3724, IHC 1:200); rabbit anti-VE-Cadherin (Cell Signaling, #2500, IHC 1:200); goat anti-mouse VE-Cadherin (R&D Systems, #AF1002, IHC 1:100); rat anti-mouse Flk1 (BD, #555307, IHC 1:100).
Corti F., Ristori E., Rivera-Molina F., Toomre D., Zhang J., Mihailovic J., Zhuang Z, & Simons M. (2022). Syndecan-2 selectively regulates VEGF-induced vascular permeability. Nature cardiovascular research, 1(5), 518-528.
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8

Antibody Panel for Vascular Signaling Analysis

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List of antibodies with application and dilutions (IHC, immunohistochemistry; WB; western blot): pVEGFR2 Y1175 (WB 1:1000, Cell Signaling #2478), pVEGFR2 Y1059 (for HUVEC, WB 1:1000, Cell Signaling #3817, ), pVEGFR2 Y1059 (for mouse ECs, WB 1:1000, EMD Millipore #ABS553), pVEGFR2 Y951 (WB 1:1000, Cell Signaling #4991 ), VEGFR2 total (WB 1:1000, Cell Signaling #2479), VE-Cad (for HUVEC, WB 1:200, Santa Cruz #sc-9989 (F-8) ), VE-Cad (for mouse ECs, WB 1:500, BD Pharmingen #555289), mouse Sdc2 (WB 1:200, R&D #AF6585 - polyclonal raised against mouse Sdc2 ED), human Sdc2 (WB 1:200, R&D # AF2965, polyclonal raised against mouse human ED), mouse PTP1b (WB 1:200, Santa Cruz #sc-1718-R (N19-R)), human PTP1b (WB 1:500, BD Pharmingen #610139), VEPTP ( VE-PTP-C pAb69 and VE-PTP 1–8 pAb70 were gifts from Prof. Dietmar Vestweber, Max Plank Institute for Molecular Biomedicine, Münster, Germany), DEP1 (WB 1:200, Santa Cruz #sc-21761 (143–41)) NRP1 (WB 1:1000, Cell signaling #3725,), HA-tag (WB 1:1000, Cell signaling #3724), rabbit anti-Rab5 (Cell Signaling, #3547, IHC 1:200); goat anti-VEGFR2 (R&D, #AF357, IHC 1:100); mouse anti-HA.11 (Covance, #MMS-101P, IHC 1:200); rabbit anti-HA (Cell signaling, #3724, IHC 1:200); rabbit anti-VE-Cadherin (Cell Signaling, #2500, IHC 1:200); goat anti-mouse VE-Cadherin (R&D Systems, #AF1002, IHC 1:100); rat anti-mouse Flk1 (BD, #555307, IHC 1:100).
Corti F., Ristori E., Rivera-Molina F., Toomre D., Zhang J., Mihailovic J., Zhuang Z, & Simons M. (2022). Syndecan-2 selectively regulates VEGF-induced vascular permeability. Nature cardiovascular research, 1(5), 518-528.
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