vegfr2 staining  (Santa Cruz Biotechnology)

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a , Quantification of 10E4, Siglec-11, 9D5, cs-DDX21 and cs-hnRNP-U intensity per cell from three independent staining experiments on HUVECs. Data are mean ± s.e.m. a.u., arbitrary units. b , Western blot analysis of whole-cell lysates isolated from HUVECs after starvation and treatment with an RNase pool followed by 3 ng ml −1 VEGF-A 165 , VEGF-A 121 or EGF stimulation (top). Quantification of the ratio of pERK to total ERK was also calculated across the biological triplicates (bottom). Statistical assessment was performed with a two-sided Student’s t -test and P values are shown. Data are mean ± s.e.m. c , Representative images of starved HUVECs treated with an RNase pool, and subsequently with VEGF-A 165 or VEGF-A 121 , finally stained with anti-VEGF-A 165 (red) or anti-VEGF-A (red). Statistical assessment was performed with a Student’s t -test and P values are shown. Data are mean ± s.e.m. NS, not significant. d , Representative images of HUVECs treated with an RNase pool and stained with anti-VEGFR2 (red). Statistical assessment was performed with a Student’s t -test and P values are shown. Data are mean ± s.e.m. e , Representative images of starved HUVECs treated with VEGF-A 165 and then co-stained with anti-VEGF-A 165 (purple) and Siglec-11 (yellow). Three independent experiments were performed. f , Schematic of the microfluidic chip (top left) used to grow HUVECs without (top middle) and with (top right) RNase A for 6 days. Representative images of BFP expressed in the HUVECs. Statistical assessment of the total migration area was performed using an unpaired two-sided Student’s t -test (bottom). Data are mean ± s.e.m. Four independent experiments were performed. g , Representative image (maximum z -projection view (left) and z -projection slice view (right)) of sprouts from a +RNase A device for the cells (blue), F-actin (red) and PECAM1 (green). Four independent experiments were performed.

Article Snippet: For VEGF-A 165 , VEGF-A 121 and VEGFR2 staining, 1 μg ml −1 of anti-VEGF-A 165 (R&D Systems), 1 μg ml −1 of anti-VEGF-A (Proteintech), and 1 μg ml −1 anti-VEGFR2 (R&D Systems) were precomplexed with 0.5 μg ml −1 of goat anti-rabbit AF647 secondary antibody and donkey anti-goat AF647 secondary antibody (Thermo Fisher Scientific).

Techniques: Staining, Western Blot, Isolation, Migration

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Western blot analysis of whole cell lysate isolated from HUVECs after starvation and treatment with RNase pool followed by 25 ng/mL VEGF-A 165 stimulation. Quantification of the ratio of phosphorylated ERK (pERK) to total ERK is calculated across the biological triplicates. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. b . Western blot analysis as in (A) with HUVECs stimulated with 25 ng/mL of VEGF-A 121 . Statistical assessment and data are as in ( a ). c . Western blot analysis as in (A) with HUVECs stimulated with 25 ng/mL of EGF. Statistical assessment and data are as in ( a ). d . Western blot analysis as in (A) with HUVECs stimulated with 3 ng/mL of VEGF-A 165 . Quantification of the ratio of phosphorylated VEGFR2 (pVEGFR2) to total VEGFR2 is calculated across the biological triplicates. Statistical assessment and data are as in ( a ). e . MST assay of VEGF-A and small RNA binding. n = 3 biologically independent repeats. Data are mean ± s.e.m. f . Representative images of HUVECs after serum starvation and treated with or without VEGF-A 165 and then co-stained with anti-VEGF-A 165 (purple) and Siglec-11 (yellow). Zoomed region shown as an inset. Scale bar, 10 µm. 3 independent experiments were performed. g . Nearest neighbor distance analysis of the VEGF-A 165 and Siglec-11 in Figure S5E. For each pair, the nm distance from VEGF-A 165 to Siglec-11 was calculated across. These values were plotted in a density histogram. h . The four biological replicate microfluidic chip without (left) and with (right) 10 µM RNase A for 6 days. Scale bar, 200 µm. i . Immunofluorescence confocal image (maximum z-projection view) of HUVEC structures after 6 days of +RNase treatment on microfluidic chip. Cells (blue), F-actin (red), and PECAM1 (green). Scale bar, 100 µm. 4 independent experiments were performed.

Article Snippet: For VEGF-A 165 , VEGF-A 121 and VEGFR2 staining, 1 μg ml −1 of anti-VEGF-A 165 (R&D Systems), 1 μg ml −1 of anti-VEGF-A (Proteintech), and 1 μg ml −1 anti-VEGFR2 (R&D Systems) were precomplexed with 0.5 μg ml −1 of goat anti-rabbit AF647 secondary antibody and donkey anti-goat AF647 secondary antibody (Thermo Fisher Scientific).

Techniques: Western Blot, Isolation, RNA Binding Assay, Staining, Immunofluorescence

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Western blot analysis of the indicated amount of VEGF-A 165 . 2 independent experiments were performed. b . Lysates from HUVECs after serum starvation, treatment with or without 25 ng/mL VEGF-A 165 , and UV-crosslinking, were treated with or without RNase pool and immunoprecipitated (IP) with an anti-VEGF-A antibody (Proteintech). Immunoprecipitated samples were analyzed by Western blot using an anti-VEGF-A 165 antibody (R&D system). 3 independent experiments were performed. c . Principal component analysis (PCA) of VEGF-A 165 RIP-seq results. d . Enriched transcripts in VEGF-A 165 RIP-seq. Each dot represents a unique small non-coding RNA (ncRNA) transcript, colored by biotype as indicated in the legend. The red dotted line marks the filtering citeria: log 2 FoldChange (IP/Input) > 0.5 and adjusted p-value < 0.05. Statistical assessment was performed with a two-sided Wald test and P values are adjusted for multiple comparisons using the Benjamini-Hochbery procedure. Solid circles indicate true positive hits that were also significantly enriched relative to the IgG control (see for details). e . Abundance of enriched transcripts grouped by biotype. Color scheme matches ( d ). f . Overlap between enriched transcripts and glycoRNA defined by ManNAz-seq, grouped by ncRNA family. The p-value was calculated using a two-sided hypergeometric test with all human small ncRNAs as the background. g . Western blot analysis of the beads pre-conjugated with 5 µg of VEGF-A 165 . h . Representative images of the indicated HUVECs stained with anti-VEGF-A 165 (red). Scale bar, 10 µm. Quantification of the images with number of cells noted per biological triplicate. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. i . Western blot analysis of whole cell lysate isolated from the indicated HUVECs. Quantification of the ratio of phosphorylated VEGFR2 (pVEGFR2) to total VEGFR2 is calculated across the biological triplicates. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. j . In vitro IP of VEGF-A 165 HS WT or HS(R/K) with small RNA and rPAL. 3 independent experiments were performed. k . MST assay of VEGF-A and small RNA treated with sialidase binding. n = 3 biologically independent repeats. Data are mean ± s.e.m. l . RNA-seq analysis of the sulfotransferases expressed from HUVECs and their FPKM values from ref. . m . EMSA analysis of the indicated VEGF-A proteins with or without the addition of rHS29.

Article Snippet: For VEGF-A 165 , VEGF-A 121 and VEGFR2 staining, 1 μg ml −1 of anti-VEGF-A 165 (R&D Systems), 1 μg ml −1 of anti-VEGF-A (Proteintech), and 1 μg ml −1 anti-VEGFR2 (R&D Systems) were precomplexed with 0.5 μg ml −1 of goat anti-rabbit AF647 secondary antibody and donkey anti-goat AF647 secondary antibody (Thermo Fisher Scientific).

Techniques: Western Blot, Immunoprecipitation, Control, Staining, Isolation, In Vitro, Binding Assay, RNA Sequencing

Journal: The American Journal of Pathology

Article Title: Activin A Prevents Hyperresponsiveness to Vascular Endothelial Growth Factor in Pathologic Blood Vessels by Perturbing the Trafficking of Activated Vascular Endothelial Growth Factor Receptor 2

doi: 10.1016/j.ajpath.2025.05.022

Figure Lengend Snippet: Activin reduces expression of Rab11. A: Diagram of VEGFR2 trafficking. Vascular endothelial growth factor (VEGF)–mediated activation of VEGFR2 triggers its internalization and trafficking. Rabs govern trafficking decisions that result in the degradation or recycling of VEGFR2. The destination of the activated VEGFR2, as well as its interaction with protein tyrosine phosphatase (PTP) 1b, influences VEGF-induced signaling. B: Confluent human retinal endothelial cells (HRECs) were treated for 24 hours with either vehicle [0.1% bovine serum albumin (BSA) in water; black bar] or activin (50 ng/mL; gray bar), harvested, and subjected to quantitative real-time PCR analysis. The resulting data were normalized to ACTB (β-actin) and expressed as relative expression. The data in the bar graph are the results of a single representative experiment. At least three independent experiments showed similar results. C: Same as B , except cells were harvested and subjected to Western blot analysis using the indicated antibodies. The images are representative Western blot analyses; the bar graphs show the mean expression in response to activin in three to five independent experiments. The molecular mass of Rab5 is 24 kDa; RABEP2, 64 kDa; Rab7, 2 kDa 2; Rab11, 25 kDa. RasGAP (124 kDa) was included as a loading control. D: Immunostaining for VEGFR2 (green) and Rab11 (red) in HRECs pretreated with activin (50 ng/mL) or vehicle for 24 hours before treatment with VEGF (100 ng/mL) or vehicle [phosphate-buffered saline (PBS)] for 30 minutes. The graph depicts the percentage of VEGFR2 that colocalized with Rab11. Activin reduced VEGFR2-Rab11 colocalization in VEGF-treated cells in two independent experiments; the same trend was observed in a third experiment, which did not reach statistical significance. The difference in means between two groups was analyzed using t -test. Data are given as means ± SEM ( B – D ). n = 4 to 6 fields of view per group. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Scale bars = 10 μm ( D ). NS, not statistically significant.

Article Snippet: Human vascular endothelial (VE)–cadherin antibody (MAB9381-SP) and VEGFR2 antibody for staining (AF357) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Control, Immunostaining, Saline

Journal: The American Journal of Pathology

Article Title: Activin A Prevents Hyperresponsiveness to Vascular Endothelial Growth Factor in Pathologic Blood Vessels by Perturbing the Trafficking of Activated Vascular Endothelial Growth Factor Receptor 2

doi: 10.1016/j.ajpath.2025.05.022

Figure Lengend Snippet: Activin suppresses VEGFR2 phosphorylation after ≥30 minutes of stimulation with vascular endothelial growth factor (VEGF). A: Cells were pretreated for 24 hours with either activin (50 ng/mL; +) or vehicle [0.1% bovine serum albumin (BSA) in water; −] and were then stimulated with phosphate-buffered saline (PBS; −) or VEGF (100 ng/mL; +) for the indicated durations. Cells were then harvested, and clarified lysates were subjected to Western blot analysis using the indicated antibodies. The phosphorylated VEGFR2 (pVEGFR2) band signal was quantified and normalized to the indicated loading control. The bar graph represents the mean ratio of normalized VEGFR2 signal in resting and VEGF-stimulated cells from at least five independent experiments. B and C: Confluent human retinal endothelial cells (HRECs) were pretreated with activin vehicle (0.1% BSA in water; −) or activin (50 ng/mL; +) for 24 hours and then stimulated with PBS (−) or VEGF (100 ng/mL; +) for the indicated duration. Because the duration of stimulation with VEGF was so long in C (6 hours), the cells were pretreated with activin for only 18 hours so that the total duration of treatment with activin at the time of harvest would match the other panels in this series of experiments. Cells were harvested, and clarified lysates were subjected to Western blot analysis using the indicated antibodies. The signal intensity of the phosphorylated Y1175 (p-Y1175) band was quantified and normalized to the loading control (β-actin or RasGAP). The bar graph shows the mean ratio of the normalized p-Y1175 signal in resting and VEGF-stimulated cells from at least three independent experiments. Data are given as means ± SEM ( A – C ). ∗ P < 0.05. NS, not statistically significant.

Article Snippet: Human vascular endothelial (VE)–cadherin antibody (MAB9381-SP) and VEGFR2 antibody for staining (AF357) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Saline, Western Blot, Control

Journal: Scientific Reports

Article Title: Multi-physics interactions drive VEGFR2 relocation on endothelial cells

doi: 10.1038/s41598-017-16786-4

Figure Lengend Snippet: VEGF-A and gremlin induce VEGFR2 rearrangement on EC surface. ( A ) ECD-VEGFR2-EYFP ECs were stimulated by a VEGF-A or gremlin gradient for 2 hours, fixed and analysed using a Zeiss Axiovert 200M system (630×; white bar: 10 μm). Arrows indicate ECD-VEGFR2-EYFP-enriched cell lamellipodia. ( B ) HUVECs adherent on Fibrinogen or VEGF-A-enriched substrates were stained for VEGFR2 (green) and actin (red) and analysed using a LSM510 Meta confocal microscope. Images show the basal portion of adherent cells with the orthogonal z reconstruction of the whole cell (630×; white bar: 10 μ m). ( C ) VEGFR2-EC, seeded on immobilized gremlin or on coverglass for 4 hours, were incubated with 150 ng/mL of gremlin for 90 minutes at 4 °C and washed with 1.5 mol/L NaCl. VEGFR2-bound gremlin, in the apical portion of the cells, was detected by immunofluorescence analysis using a Zeiss Axiovert 200 M microscope system (630x; white bar: 10 μ m). Data are expressed as percentage ± s.d. of gremlin positive area with respect to the total cell area (n = 20 cells/sample; * P < 0.001, Student’s t-test).

Article Snippet: For total VEGFR2 staining, samples were incubated overnight with a rabbit polyclonal anti-VEGFR2 antibody (1:200, Santa Cruz Biotechnology) followed by a 1-hour incubation with AlexaFluor 488-conjugated anti-rabbit IgG (1:500).

Techniques: Staining, Microscopy, Incubation, Immunofluorescence

Journal: Scientific Reports

Article Title: Multi-physics interactions drive VEGFR2 relocation on endothelial cells

doi: 10.1038/s41598-017-16786-4

Figure Lengend Snippet: VEGF-A and gremlin reduce VEGFR2 motility on EC surface. ( A ) FRAP analysis was performed on cell plasma membrane of serum-starved ECD-VEGFR2-EYFP over-expressing GM7373 cells treated or not with VEGF-A or gremlin (50 ng/mL). Images were acquired at one per minute for 12 minutes, 2 before and 10 after bleaching. The bleached area is indicated by a square and the recovery time is indicated over the images as seconds after photobleaching (630x; white bar: 20 μ m). ( B ) Collected images were analyzed using simFRAP ImageJ plugin to calculate diffusion coefficients. The graph shows diffusion coefficient mean ± s.d. of control, VEGF-A, and gremlin treated cells ( n = 7 – 15; * P < 0.01, Student’s t-test).

Article Snippet: For total VEGFR2 staining, samples were incubated overnight with a rabbit polyclonal anti-VEGFR2 antibody (1:200, Santa Cruz Biotechnology) followed by a 1-hour incubation with AlexaFluor 488-conjugated anti-rabbit IgG (1:500).

Techniques: Clinical Proteomics, Membrane, Expressing, Diffusion-based Assay, Control

Journal: Scientific Reports

Article Title: Multi-physics interactions drive VEGFR2 relocation on endothelial cells

doi: 10.1038/s41598-017-16786-4

Figure Lengend Snippet: Time evolution of the VEGFR2-gremlin complex formation on the EC membrane. Comparison is made between the VEGFR2 total fluorescence intensity (free and bound) in contact the substrate (red dots) and the numerical simulation data (green lines). To allow comparisons, both sets of data have been normalized to the values reached at the final time t F = 7200 s.

Article Snippet: For total VEGFR2 staining, samples were incubated overnight with a rabbit polyclonal anti-VEGFR2 antibody (1:200, Santa Cruz Biotechnology) followed by a 1-hour incubation with AlexaFluor 488-conjugated anti-rabbit IgG (1:500).

Techniques: Membrane, Comparison, Fluorescence

Journal: Scientific Reports

Article Title: Multi-physics interactions drive VEGFR2 relocation on endothelial cells

doi: 10.1038/s41598-017-16786-4

Figure Lengend Snippet: The chemo-mechanical transport model describes VEGFR2 relocation dynamics. ( A ) Time evolution of the spatial concentration c R of free VEGFR2 along the cell membrane. Each curve plots the distribution of free receptors at different times t = 60 n , with n = 0, 1, 2, … 120 s from the beginning of the experiment at t = 0 to the final time t F = 7200 s. ( B ) Spatial evolution of the concentration c C of the receptor-ligand complex at various times. The curves report the numerical simulation: points A, B, and C correspond to those in ( A ). ( C ) VPM staining for VEGFR2 confirms peaks in the intensity of fluorescence at the boundary of the substrate/membrane contact surface.

Article Snippet: For total VEGFR2 staining, samples were incubated overnight with a rabbit polyclonal anti-VEGFR2 antibody (1:200, Santa Cruz Biotechnology) followed by a 1-hour incubation with AlexaFluor 488-conjugated anti-rabbit IgG (1:500).

Techniques: Concentration Assay, Membrane, Staining, Fluorescence