Journal: bioRxiv

Article Title: Inhibition of peripheral VEGF signaling rapidly reduces leucocyte obstructions in brain capillaries and increases cortical blood flow in an Alzheimer’s disease mouse model

doi: 10.1101/2021.03.05.433976

Figure Lengend Snippet: ELISA measurements of VEGF1R receptor ( A ), VEGF2R receptor ( B ) from brain lysates, and soluble VEGF1R receptor ( C ), and soluble VEGF2R receptor ( D ) from plasma samples after 1 week of anti-VEGF-A treatment or saline injections in APP/PS1 and WT mice (n=3 mice per group). Kruskal-Wallis test with multiple comparison correction to compare across groups. In all graphs each point represents the ELISA measurement from one mouse, red horizontal line represents the median; * indicates p = 0.05.

Article Snippet: After the measuring the concentrations ELISAs were performed following the manufacture’s protocol (VEGF1R (MVR100) and VEGF2R (MVR200B), R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Saline, Comparison

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: HBMECs were starved with low-serum medium for 6 hours and stimulated with growth factors at the indicated concentrations for 30 min (VEGFR1) or 10 min (VEGFR2). The receptor activation was picked up either by ELISA detecting the pan-tyrosine phosphorylation of VEGFR1 (A–C) or VEGFR2 (D) , or by immunoblotting and probed with primary antibody targeting VEGFR2 phosphorylation at tyrosine site 1175 (E) .

Article Snippet: After being briefly vortexed and centrifuged at 12,000× g for 10 min at 4 °, the cell lysates went through the protocol of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170), Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766), or Human Phospho-PDGFRβ DuoSet IC ELISA kit (R&D Systems, DYC1767) to assess the phosphorylation levels of VEGFR1, VEGFR2, or PDGFRβ, respectively.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: (A– G) HDMEC ( PDGFRA −/− and PDGFRB −/− ) were starved with low-serum medium for 6 hours and stimulated with growth factors at the indicated concentrations for 30 min (VEGFR1) or 10 min (VEGFR2). (A–C) The fold changes of VEGFR1 phosphorylation induced were assessed via ELISA. (D–G) The fold changes of VEGFR1 phosphorylation induced were assessed via immunoblotting. (H) The fold changes of PDGFRβ phosphorylation in HDFs induced by 30-min VEGF-A or PDGF-BB treatment were measured via ELISA.

Article Snippet: After being briefly vortexed and centrifuged at 12,000× g for 10 min at 4 °, the cell lysates went through the protocol of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170), Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766), or Human Phospho-PDGFRβ DuoSet IC ELISA kit (R&D Systems, DYC1767) to assess the phosphorylation levels of VEGFR1, VEGFR2, or PDGFRβ, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The docking protein FRS2α is a critical regulator of VEGF receptors signaling

doi: 10.1073/pnas.1404545111

Figure Lengend Snippet: FRS2α knockdown in HUVEC inhibits VEGF-A165–dependent signaling. (A) Control and FRS2α knockdown HUVEC were serum starved overnight and treated with VEGF-A165 (50 ng/mL) as indicated. Cell lysates were immunoprecipitated (IP) with an anti-VEGFR2 antibody and immunoblotted with anti–p-Tyrosine antibody. The same blot was stripped and blotted with anti-VEGFR2. Input lysates were blotted with anti-VEGFR2 and anti-FRS2α antibodies. (B) Control and FRS2α knockdown HUVEC were serum starved overnight and treated with VEGF-A165 (50 ng/mL) as indicated. Cell lysates were blotted with p-VEGFR2, VEGFR2, p-ERK, ERK, and FRS2α antibodies. (C) Control and FRS2α-6F (Flag) overexpressed HUVEC were serum starved overnight and treated with VEGF-A165 (50 ng/mL). Cell lysates were blotted with p-VEGFR2, VEGFR2, p-ERK, ERK, and Flag (FRS2α) antibodies. (D) Control and FRS2α knockdown HUVEC were serum starved overnight. Cell proliferation (Left) and cell migration (Right) in response to VEGF-A165 (50 ng/mL) profiles are shown as detected by xCELLigence. Approximately 1000 cells (for cell proliferation) or 25,000 cells (for cell migration) were loaded per well in duplicate (***P < 0.01 compared with control). (E) In vitro Matrigel: The extent of cords branching was assessed in control and FRS2α knockdown HUVEC placed on growth factor-depleted Matrigel and exposed to VEGF-A165 (50 ng/mL). Data in A–C and E are based three independent experiments; data in D are based on two independent experiments.

Article Snippet: The following antibodies were used for immunoblotting (IB), immunoprecipitation (IP), or immunohistochemistry (IHC): CD31 (ab28364, Abcam; IHC), CD31 (553370, BD Pharmingen; EC isolation), p-ERK (M8159, Sigma; IB), FLAG (F1804, Sigma; IB), FRS2α (H-91, Santa Cruz; IB), p44/p42 MAP Kinase (9102, Cell Signaling; IB), Phosphotyrosine, clone 4G10 (05–321, Millipore; IB), Phosphotyrosine (PY20) (sc-508, Santa Cruz; IB), β-tubulin (T7816, Sigma; IB), VEGFR2 (2479, Cell Signaling; IB), VEGFR2 (5168, Cell Signaling; IP), p-VEGFR2 Tyr-801 (VP2921, ECM Biosciences; IB), p-VEGFR2 Tyr-951 (4991, Cell Signaling; IB), p-VEGFR2 Tyr-1054/1059 (441047G, Invitrogen; IB), p-VEGFR2 Tyr-1175 (2478, Cell Signaling; IB), and p-VEGFR2 Tyr-1214 (44-1052, Invitrogen; IB).

Techniques: Knockdown, Control, Immunoprecipitation, Migration, In Vitro