Journal: The Journal of Cell Biology

Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

doi: 10.1083/jcb.200608093

Figure Lengend Snippet: MSCs expressed no VEGFRs. The expression of VEGFR mRNA transcripts was examined by semiquantitative RT-PCR analysis and cell surface protein expression by single-color flow cytometry, using human MSCs, HUVECs as a VEGFR-positive control cell, and HDF as a VEGFR-negative control cell. (A) RNA isolated from MSCs, HUVECs, and HDFs were used to amplify VEGFR1-3, VEGF-A, NP-1, and NP-2 transcripts, with GAPDH as a control, and then resolved by agarose gel. Lane 1, VEGFR1 (99 bp); lane 2, VEGFR2 (81 bp); lane 3, VEGFR3 (87 bp); lane 4, VEGF-A (98 bp); lane 5, GAPDH (71 bp); lane 6, NP-1 (77 bp); lane 7, NP-2 (83 bp). Data are representative of six independent experiments, with two different pairs of primers for VEGFR1-3 used. (B) Flow cytometry using PE-conjugated antibodies. Analysis of VEGFR1-3 is represented by VEGFR1-, VEGFR2-, and VEGFR3-PE expression, respectively, with IgG 1 -PE expression as a control. A representative example of three independent experiments is shown.

Article Snippet: For single-color flow cytometry, MSCs, HUVECs, or HDFs (4×10 6 cells/ml) were incubated with either PE-conjugated anti–human VEGFR1-PE (FAB321P), VEGFR2-PE (FAB357P), or VEGFR3-PE (FAB3492P) antibodies, or control anti–IgG 1 -PE antibody (IC002P; R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Positive Control, Negative Control, Isolation, Control, Agarose Gel Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

doi: 10.1083/jcb.200608093

Figure Lengend Snippet: VEGF-A stimulated both PDGFRα and PDGFRβ tyrosine phosphorylation. Human phospho-RTK arrays were used to examine VEGF-A–induced RTK phosphorylation levels in MSC lysate samples. Arrays contain phosphotyrosine-positive control spots in each corner, having coordinates (A1, A2), (A23, A24), (F1, F2), (F23, F24), which were assigned a pixel density value of 100, which was used to normalize positive RTK spot pixel density values. Relevant RTK duplicate spot coordinates: PDGFRα = (C7, C8), PDGFRβ = (C9, C10), VEGFR1 = (D9, D10), VEGFR2 = (D11, D12), VEGFR3 = (D13, D14), EGFR = (B1, B2), FGFR3 = (B13, B14), Axl = (B21, B22), EphA7 = (E3, E4). (A) RTK array analysis of control MSC lysate, not stimulated with exogenous growth factor (basal). (B) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml VEGF-A 165 in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. (C) Bar graph representing data from arrays (A and B). Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. A representative example of two independent experiments is shown for each array analysis. *, P < 0.001 compared with the respective VEGF-A 165 –stimulated scrambled siRNA control.

Article Snippet: For single-color flow cytometry, MSCs, HUVECs, or HDFs (4×10 6 cells/ml) were incubated with either PE-conjugated anti–human VEGFR1-PE (FAB321P), VEGFR2-PE (FAB357P), or VEGFR3-PE (FAB3492P) antibodies, or control anti–IgG 1 -PE antibody (IC002P; R&D Systems).

Techniques: Phospho-proteomics, Positive Control, Control, Transfection

Journal: Clinical Cancer Research

Article Title: Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR-3 Are Localized Primarily to the Vasculature in Human Primary Solid Cancers

doi: 10.1158/1078-0432.ccr-09-2797

Figure Lengend Snippet: Fig. 1. Specificity of VEGFR-2 and VEGFR-3 antibodies. A, the specificity of antibodies raised to VEGFR-2 (55B11, sc-315, sc-504, and sc-6251) and VEGFR-3 (AF349) was determined by Western blot analysis of protein extracts from PAE and NIH-3T3 cells engineered to overexpress VEGFR-1, VEGFR-2, VEGFR-3, and CSF1R and cells expressing endogenous PDGFR-β (VSMC) and c-Kit (M-07e). B, 55B11 and AF349 were evaluated by immunocytochemical analysis of FFPE PAE cells engineered to overexpress VEGFRs.

Article Snippet: To do this, we determined the crossreactivity of human VEGFR antibodies using multiple www.aacrjournals.org Researc on Januarclincancerres.aacrjournals.org Downloaded from screening platforms and identified two antibodies, 55B11 (VEGFR-2; Cell Signaling Technology) and AF349 (VEGFR-3; R&D Systems), with credible specificity.

Techniques: Western Blot, Expressing

Journal: Circulation

Article Title: Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension

doi: 10.1161/circulationaha.116.025390

Figure Lengend Snippet: Figure 2. VEGFR3 and BMPR2 co-internalize upon ligand stimulation

Article Snippet: For VEGFR3 expression lentivirus, VEGFR3 construct was purchased from Origene Technologies and cloned into pCDH-EF1-MCS lentivirus backbone vector (System Biosciences) to generate pCDH-EF1-hVEGFR3 vector.

Techniques:

Journal: Circulation

Article Title: Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension

doi: 10.1161/circulationaha.116.025390

Figure Lengend Snippet: Figure 2C). * P < 0.05 Both BMPR2 and VEGFR3 appear to undergo endocytosis upon BMP6

Article Snippet: For VEGFR3 expression lentivirus, VEGFR3 construct was purchased from Origene Technologies and cloned into pCDH-EF1-MCS lentivirus backbone vector (System Biosciences) to generate pCDH-EF1-hVEGFR3 vector.

Techniques: