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

Journal: Animals : an Open Access Journal from MDPI

Article Title: Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct

doi: 10.3390/ani11041099

Figure Lengend Snippet: Primer sequences used for gene transcription analysis by real-time PCR.

Article Snippet: The relative protein abundance of FGFR1, FGFR2 and KDR was measured using specific primary antibodies against FGFR1 (Orb 156864, Biorbyt, Cambridge, UK, dilution 1:500), FGFR2 (SC 6930, Santa Cruz Biotechnology, Dallas, USA, dilution 1:500) and KDR (Orb 99143, Biorbyt, Cambridge, UK, dilution 1:250).

Techniques: Sequencing, Amplification

Journal: Animals : an Open Access Journal from MDPI

Article Title: Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct

doi: 10.3390/ani11041099

Figure Lengend Snippet: Relative quantification of FGFR1 , FGFR2 and KDR transcripts in the equine oviduct ( n = 5 samples for each estrous cycle phase; n = 5 for each portion of oviduct analyzed). Comparison of transcripts between the follicular phase, early-luteal phase (Early LP), and mid-luteal (Mid LP) ( A , C , E ). Comparison of transcripts between oviduct portions (infundibulum, ampulla, isthmus), regardless of the phase of the estrous cycle ( B , D , F ). Bars represent mean ± SEM. AU: arbitrary units. Different letters indicate significant differences ( p < 0.05).

Article Snippet: The relative protein abundance of FGFR1, FGFR2 and KDR was measured using specific primary antibodies against FGFR1 (Orb 156864, Biorbyt, Cambridge, UK, dilution 1:500), FGFR2 (SC 6930, Santa Cruz Biotechnology, Dallas, USA, dilution 1:500) and KDR (Orb 99143, Biorbyt, Cambridge, UK, dilution 1:250).

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct

doi: 10.3390/ani11041099

Figure Lengend Snippet: Relative protein abundance of FGFR1, FGFR2 and KDR in the equine oviduct ( n = 5 samples for each estrous cycle phase; n = 5 for each portion of oviduct analyzed). Comparison of relative protein abundance between oviduct portions (infundibulum, ampulla, isthmus), regardless of the phase of the estrous cycle ( A , C , D ), and their respective representative Western blot bands ( B , D , F ). Bars represent mean ± SEM. AU: arbitrary units. Different letters indicate significant differences ( p < 0.05).

Article Snippet: The relative protein abundance of FGFR1, FGFR2 and KDR was measured using specific primary antibodies against FGFR1 (Orb 156864, Biorbyt, Cambridge, UK, dilution 1:500), FGFR2 (SC 6930, Santa Cruz Biotechnology, Dallas, USA, dilution 1:500) and KDR (Orb 99143, Biorbyt, Cambridge, UK, dilution 1:250).

Techniques: Western Blot