fgfr2α iiic fc  (R&D Systems)

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 activates a previously unidentified protein in endothelial cells. A HUVEC were incubated for 5 minutes with Ang2 (100, 500 and 1000 ng/ml with or without sodium orthovanadate (Na 3 VO 4 ) after overnight starvation. None: HUVEC cultured under standard conditions. Cell lysates were tested for p-EphrinB T324/329 , EphrinB2, p-TIE2, and ß-actin. After 5 minutes incubation with Ang2, a ~130kDa band is recognized by p-EphrinB but not by EphrinB2 or p-TIE2 antibodies. B Time dependent decrease in intensity of the ~130kDa band identified by p-EphrinB antibodies. Relative band intensity was measured by ImageJ. C Experimental design for proximity labeling. Starved HUVEC were incubated for 5 minutes or 6 hours with or without His-tag Ang2 (100 ng/ml). After crosslinking (2 mM DSP), incubation with the primary anti-His-tag antibody, addition of HRP-conjugated secondary antibody plus hydrogen peroxide and phenol biotin, protein purification with streptavidin-coated Dynabeads, the eluted proteins were analyzed by LC-MS-MS. D Relative abundance of selected proteins identified by LC-MS-MS in HUVEC incubated for 5 minutes with medium only or with Ang2. FGFR2 is identified among the most abundant proteins specifically associated with Ang2 in HUVEC by proximity labeling. The results are evaluated by model-based analysis of proteomic data (MAP). E Relative abundance of selected Ang2-associated proteins detected by proximity labeling in HUVEC incubated for 6 hours with Ang2. F Venn diagram depicts the number of Ang2-interacting proteins unique to or common to the 5 minutes and 6 hours incubation. G Pathway enrichment analysis applied to the 368 Ang2-proximal proteins identified in HUVEC after 5 minutes incubation with Ang2

Article Snippet: Binding of Ang2 to FGFR2ß (IIIb)-Fc (R&D Systems 665-FR), to FGFR2α (IIIc)-Fc (R&D Systems, No. 712-FR) or hIgG-Fc (R&D Systems, No. 110-HG) was assessed using the previously described binding buffer [ ] for 1 h. Immunoprecipitation was performed using proteinG-coated Dynabeads (ThermoFisher Scientific, No. 10004D) or with NTA-coated Dynabeads for His-Tag protein pulldown (ThermoFisher Scientific, No. 10103D).

Techniques: Incubation, Cell Culture, Labeling, Protein Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Recombinant Ang2 binds to recombinant FGFR2-Fc. A Lysates prepared from HUVEC in starvation medium for 1 hour and then incubated with FGF1 (5 nM, 5 min) or/and Ang2 (5 nM, 5 min) in the same medium were analyzed by Western blotting; antibodies to p-FGFR Y653/654 and FGFR2 recognize a band at ~130 kDa. Representative of 3 experiments. B Schematic of the pull-down experiment. FGFR2: FGFR2ß (IIIb)-Fc. C Ang2 (0.1 μM) specifically binds to recombinant FGFR2ß (IIIb)-Fc (0.1-0.5 μM) but not to human IgG-Fc (Fc, 0.5 μM). The precipitated proteins were immunoblotted with antibodies to Ang2 (left) or to FGFR2ß (IIIb) (right). The asterisks point to bands specifically identifying Ang2 (left) and FGFR2 (right). Representative of 3 experiments. D Schematic of the pull-down experiment. FGFR2: FGFR2α (IIIc)-Fc. E Ang2 (0.1 μM) does not compete with the binding of FGF1 (0.1-0.4 μM) to FGFR2α (IIIc)-Fc (0.2 μM). IgG-Fc used at 0.2 mM. The precipitates were immunoblotted with antibodies to Ang2, FGF1, or anti-human Fc (hFc). The asterisks point to Ang2 (left), FGF1 (middle), and to FGFR2-Fc or Fc (right). Representative of 3 experiments. F Structural modeling of the Ang2, FGF1 and FGFR2α (IIIb) trimeric complex from amino acid sequences by AlphaFold2-Multimer. Red: predicted structure of Ang2 in the predicted trimeric complex. Pink: Ang2 structure from the crystal structure of Ang2 alone (PDB 1z3s). Blue: predicted structure of FGFR2α (IIIb). Light blue: FGFR2ß (IIIb) structure from the crystal structure of FGFR2ß (IIIb)-FGF1 dimeric complex (PDB 1djs). Yellow: predicted structure of FGF1. Green: FGF1 structure from the crystal structure of FGFR2ß (IIIb)-FGF1 complex (PDB 1djs). The three predicted protein structures are largely consistent when superimposed on the respective crystal structures. The predicted structure suggests that Ang2 and FGF1 interact with FGFR2 at distinct sites. G Structural models of the Ang2, FGF1 and FGFR2α (IIIc) trimeric complex (left) and the Ang2, FGF2 and FGFR2α (IIIc) trimeric complex (right) from amino acid sequences by AlphaFold3

Article Snippet: Binding of Ang2 to FGFR2ß (IIIb)-Fc (R&D Systems 665-FR), to FGFR2α (IIIc)-Fc (R&D Systems, No. 712-FR) or hIgG-Fc (R&D Systems, No. 110-HG) was assessed using the previously described binding buffer [ ] for 1 h. Immunoprecipitation was performed using proteinG-coated Dynabeads (ThermoFisher Scientific, No. 10004D) or with NTA-coated Dynabeads for His-Tag protein pulldown (ThermoFisher Scientific, No. 10103D).

Techniques: Recombinant, Incubation, Western Blot, Binding Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: The Ang2 inhibitor AMG386 reduces Ang2 binding to FGFR2-Fc. A Schematic of the pull-down experiment. FGFR2α (IIIc)-Fc. B AMG386 (AMG, 0.2 or 0.05 μM) inhibits the binding of Ang2 (0.1 μM) to FGFR2-Fc (0.2 μM). IgG-Fc (Fc, 0.2 μM). The precipitates were immunoblotted with antibodies to Fc and Ang2. The black asterisk points to FGFR2-Fc (left) and Ang2 (right); the red asterisk points to AMG386. Representative experiment (of three performed). C Overall complex structure of AMG386 (yellow) bound to Ang2 (blue), FGF1 (green), and FGFR2α (IIIb) (pink) is shown as a surface representation. AMG386 occupies the main binding cavity of the complex, highlighting its central role in coordinating interactions with the other proteins. D and E Trp 280 from AMG386 (yellow) forms hydrophobic interactions with nearby residues, including Tyr 135 and His 108 from FGFR2 (pink) and Phe 190 from Ang2 (blue). These interactions include π-stacking between Trp 280 (AMG386) and Phe 190 (Ang2), contributing to the stabilization of the complex. Additional π-π stacking interactions involving His 108 (FGFR2) and Phe 190 (Ang2) underline the importance of aromatic side chains in maintaining the structural integrity of the complex. F A hydrogen bond between Lys 176 (FGFR2, pink) and Glu 274 (AMG386, yellow) reinforces the binding interface. G Another hydrogen bond is observed between Glu 283 (AMG386, yellow) and Gly 200 (Ang2, blue), further enhancing the specificity and stability of the interaction

Article Snippet: Binding of Ang2 to FGFR2ß (IIIb)-Fc (R&D Systems 665-FR), to FGFR2α (IIIc)-Fc (R&D Systems, No. 712-FR) or hIgG-Fc (R&D Systems, No. 110-HG) was assessed using the previously described binding buffer [ ] for 1 h. Immunoprecipitation was performed using proteinG-coated Dynabeads (ThermoFisher Scientific, No. 10004D) or with NTA-coated Dynabeads for His-Tag protein pulldown (ThermoFisher Scientific, No. 10103D).

Techniques: Binding Assay