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: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Volcano plot of proteomic analysis of murine plasma from WT and B2KO mice ( n = 6). VEGFR3 (FLT4) is highlighted in red. ( B ) Normalized VEGFR3 LFQ intensities extracted from A . ( C ) MSD-assay quantifications of sVEGFR3 in the same plasma samples. ( D ) Immunoblot detection of sVEGFR3 ectodomain in mouse plasma from A , using nonreducing and reducing conditions. ( E ) Volcano plot of proteomic analysis of murine plasma from an independent B2KO line ( n = 9) compared with WT ( n =9) and ( F ) the extracted normalized LFQ values. Volcano plots of the proteomic analyses of Bace1/Bace2 double-knockout (BDKO) mice ( n =9) compared with the WT line ( n = 9) ( G ) (corresponding extracted LFQ intensities of sVEGFR3 in F ) and B1KO ( n = 9) compared with an individual control WT line ( n = 9) ( H ). ( I ) Normalized LFQ values extracted from H . ( J ) Localization of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The signal peptide is shown in rose, the ectodomain is indicated in blue, the intracellular domain in green, and the transmembrane domain in yellow. Two sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots ( A , E , G , and H ). Proteins with P < 0.05 are shown as red circles. Extracted LFQ quantifications ( B , F , and I ) of VEGFR3 with significance after FDR correction are labeled with plus signs. All dot plots were normalized on the WT mean and depict mean and SD. MSD-assay data ( C ) additionally depicts the P value calculated by unpaired t test. **** P < 0.0001.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Western Blot, Double Knockout, Control, Sequencing, Labeling

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Schematic of VEGFR3 fragments. From left to right: The immature proVEGFR3 (200 kDa) can be cleaved by BACE2, releasing the immature, soluble ectodomain sol proVEGFR3 (130 kDa). The mature protein consists of 2 subunits linked through a disulfide bridge: VEGFR3α (75 kDa) and VEGFR3β (125 kDa). Upon BACE2 cleavage, the VEGFR3β-CTF (70 kDa) and sVEGFR3 (130 kDa) are generated, the latter of which consists of the VEGFR3α (75 kDa) and VEGFR3β-NTF (55 kDa) fragments. ( B ) Immunoblot detection of VEGFR3 in lysates and media of HEK293 cells transfected with empty control plasmids (Ctrl), Vegfr3 , Vegfr3 + Bace2 , and Bace2 . B2, BACE2. Data show 3 independent experiments. sVEGFR3 is not detectable under reducing conditions. sol proVEGFR3 in the lysates appears at around 100 kDa and derives from BACE2 cleavage of immaturely glycosylated proVEGFR3 early in the secretory pathway upon BACE2 overexpression. ( C ) Localization and length of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The ectodomain is indicated in blue, the intracellular domain in green, the signal peptide in orange, and the transmembrane domain in yellow. ( D ) N-terminal juxtamembrane region of VEGFR3 sequence. The identified semispecific peptide after LysN digestion is marked in yellow, the proposed cleavage site after amino acid alanine with 2 vertical lines, and the transmembrane region in gray. ( E ) Comparison of the fragment ion spectra of the identified C-terminal peptide of the LysN digestion KGC(cam)VN(+1)SSASVA (lower spectrum) to a synthetic peptide with the same sequence (upper spectrum). Identified y-ions are indicated in red, b-ions in blue, and fragment ions with neutral losses in green. Both spectra match with a dot product of 0.93 for the fragment ion intensities.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Generated, Western Blot, Transfection, Control, Over Expression, Sequencing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Immunoblot detection after control treatment (–) or upon BACE1 and BACE2 knockdown (+). Lysates were blotted for VEGFR3, BACE1/2, and actin. Conditioned media were blotted for sVEGFR3. ( B ) Corresponding densitometric quantifications, deriving from VEGFR3β (lysate) and sVEGFR3 (medium). ( C ) Immunoblots of cells treated with DMSO (–) or 100 nM verubecestat (+). ( D ) Corresponding densitometric quantifications as in B . Dot plots were normalized on the control mean and depict mean and SD, alongside the calculated P values, calculated by unpaired t test. ** P < 0.01; **** P < 0.0001. P values are only indicated where significance was observed. Data are derived from n = 6 biological replicates. Shown are representative data from 3 independent experiments.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Western Blot, Control, Knockdown, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Schematic for VEGFR3 signaling. Upon ligand binding, VEGFR3 dimerizes, resulting in intracellular autophosphorylation and activation of the downstream genes FOXC2 and DLL4 . V, verubecestat, inhibitor of BACE2. Gene expression levels of ( B ) DLL4 and FOXC2 and ( C ) VEGFR3 after the application of DMSO, 100 nM verubecestat (V), and VEGF-C. ( D and E ) Gene expression levels of DLL4 , FOXC2 , VEGFR3 , BACE1 , and BACE2 after BACE knockdown (siB1/siB2) without or with (+V) subsequent verubecestat application. All dot plots were normalized on the control mean and depict mean and SD alongside the P values calculated by unpaired t tests against the DMSO control ( B and C ) or by 1-way ANOVA ( D and E ), in both cases followed by Bonferroni’s multiple-comparison test. * P < 0.05; ** P < 0.01; **** P < 0.0001. P values are only indicated where significance was observed. In B , 1 data point was excluded from the DLL4 expression/VEGF-C156S data set, since it was identified as an outlier via the ROUT method. Data are derived from n = 6 ( B and C ) or n = 4 ( D and E ) biological replicates.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Ligand Binding Assay, Activation Assay, Expressing, Knockdown, Control, Comparison, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: Volcano plots of proteomic analysis of murine plasma from ( A ) compound 89–treated (Cpd89) versus vehicle-treated (veh) mice and ( B ) LY2811376-treated versus vehicle-treated mice ( n = 13, treated; n = 14, veh). VEGFR3 is highlighted in red. ( C ) Corresponding extracted LFQ intensities of sVEGFR3 and ( D ) MSD-assay quantifications of sVEGFR3. Plasma sVEGFR3 ( E ) and plasma sSEZ6L ( F ) levels in 8–10 B1KO, B2KO, and respective WT mice with (blue) or without (black) 3 days of 50 mg/kg per os twice a day verubecestat dosing. ( G ) Plasma levels of VEGFR3 and SEZ6L during 7 days of 0.1% dietary verubecestat (average drug intake, 97 mg/kg/d; n = 6 per group, all male, age: 7–10 weeks), respective to untreated control levels. Two-sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots ( A and B ). Proteins with P < 0.05 are shown as red circles. ( C ) Significance after FDR correction is indicated with plus signs. All dot plots were normalized on the control mean and depict the SD alongside the calculated P values, calculated by 1-way ( D and G ) or 2-way ( E and F ) ANOVA with Bonferroni’s multiple-comparison test. * P < 0.05; *** P < 0.001; **** P < 0.0001. P values are only indicated where significance could be observed. Number of biological replicates in E and F was 9, except for Bace1-WT + verubecestat ( n = 8) and for Bace2WT without verubecestat ( n = 10).

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Control, Comparison