anti vegf a 165  (R&D Systems)

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: Anti-VEGF-A 165 (AF293-NA, R&D Systems; immunoblot 1:1,000) and donkey anti-goat IgG secondary antibody (92632214, LI-COR Biosciences; immunoblot 1:1,000) were used as primary and secondary antibodies, separately.

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: Anti-VEGF-A 165 (AF293-NA, R&D Systems; immunoblot 1:1,000) and donkey anti-goat IgG secondary antibody (92632214, LI-COR Biosciences; immunoblot 1:1,000) were used as primary and secondary antibodies, separately.

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: Anti-VEGF-A 165 (AF293-NA, R&D Systems; immunoblot 1:1,000) and donkey anti-goat IgG secondary antibody (92632214, LI-COR Biosciences; immunoblot 1:1,000) were used as primary and secondary antibodies, separately.

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

Journal: Biomaterials advances

Article Title: Enhancing BMP-2-mediated osteogenesis with a synthetic heparan sulfate mimetic.

doi: 10.1016/j.bioadv.2023.213671

Figure Lengend Snippet: Fig. 2. Glycomimetic 15 binds BMP-2 and several other pro-regenerative factors with high affinity. A – SPR solution competition dosing experiments were performed with Ai - BMP-2, Aii - BMP-4, Aiii - BMP-7, Aiv BMPRII Fc chimera and Av - VEGF165 using a dose range of glycomimetic 15 (3.91 to 8000 nM). Avi – normalized response data extrapolated from sensorgrams Ai-Av. B – fluorescence polarization solution competition with BMP-2 (50 nM), 488-labelled heparin dp24 (0.5 nM) and increasing concentrations of glycomimetic 15 (red) or glycomimetic 18 (blue). Ci – DSF melt curves. Cii – first derivative of melt curve data. Ciii – BMP-2 melt temperature when incubated with multiple concentrations of glycomimetic 15, derived from the first derivative of melt curve data. Di – Normalized anti-factor Xa activity for glycomimetic 15 (black), heparin dp12 (red) and heparin dp24 (blue). Dii – anti-factor Xa activity in IU/mg, calculated using USP heparin standard curve. Bars represent mean, and error bars represent S.D. All data is representative of three independent experiments.

Article Snippet: BMP-2 (cat. # 355-BM/ CF), BMP-4 (cat. # 314-BP/CF), BMP-7 (cat. # 354-BP/CF), BMPRII Fc chimera (cat. # 811-BR), Noggin (cat. # 6057-NG/CF), VEGF165 (cat. # 293-VE/CF) polyclonal Goat anti-Human BMP-2/BMP-4 IgG (cat. # AF355), biotinylated monoclonal Mouse anti-Human BMP-2 IgG (cat. #BAM3552) were purchased from R&D systems.

Techniques: Fluorescence, Incubation, Derivative Assay, Activity Assay