human vegf d  (Sino Biological)

Journal: bioRxiv

Article Title: Regulation of angiogenesis by signal sequence-derived peptides

doi: 10.1101/2024.08.22.609269

Figure Lengend Snippet: The DCBLD2 SS traC subdomain is involved in DCBLD2 interaction with VEGFR2. (A) Schematic representation of constructs I-VIII, each with various combinations of DCBLD2 domains tagged with HA (I-V), DCBLD2, or Shrew-1 SS fused with GFP and HA tags (VI, VII) or GFP-HA alone (VIII). (B) Co-immunoprecipitation of the products of constructs I-V with VEGFR2 following transfection of each construct with VEGFR2 (or transfection of construct I alone without VEGFR2) in HEK 293T cells. VEGFR2 immunoprecipitation is followed by Western blotting for HA and VEGFR2. (C) Western blot analysis of the effects of constructs I-V on VEGF-induced ERK phosphorylation following their transfection in Dcbld2 -/- murine lung endothelial cells transfected. (D) Co-immunoprecipitation of the products of constructs VI-VIII with VEGFR2 following their transfection in HEK 293T cells. HA immunoprecipitation is followed by Western blotting for HA and VEGFR2. (E) NtraC model of DCBLD2 SS. (F) Schematic representation of constructs IX-XI encompassing different DCBLD2 SS subdomains fused with GFP and HA tags (IX-XI) or GFP-HA alone (VIII). (G) Co-immunoprecipitation of the products of constructs IX-XI with VEGFR2 following their transfection in HEK 293T cells. HA immunoprecipitation is followed by Western blotting for HA and VEGFR2. SS: signal sequence; TMD: transmembrane domain. HA: Hemagglutinin; GFP: green fluorescent protein; IP: immunoprecipitation; WB: western blot; WT: wild type.

Article Snippet: Subsequently, either 4 µl of FITC-traC (0.75 µg/µl), 4 µl of recombinant human VEGF (1 µg/µl, R&D Systems), or 4 µl of a solution containing both FITC-traC (0.75 µg/µl) and VEGF (1 µg/µl) was added and mixed.

Techniques: Construct, Immunoprecipitation, Transfection, Western Blot, Sequencing

Journal: bioRxiv

Article Title: Regulation of angiogenesis by signal sequence-derived peptides

doi: 10.1101/2024.08.22.609269

Figure Lengend Snippet: The DCBLD2 SS FITC-traC peptide promotes VEGF and ischemia-induced angiogenesis in vivo. (A, B) Illustrative photographs (A) and CD31 immunofluorescent staining (red, n=5, B) of matrigel plugs with or without VEGF and FITC-traC, collected after 5 days post-implantation. Nuclei are stained blue with DAPI. Bar = 200μm. (C, D) Illustrative slit-lamp photographs (C) and CD31 immunofluorescent staining (red, n=10, D) of corneas implanted with sustained- release pellets containing either FITC-traC or DMSO and with or without VEGF. Bar= 1mm. (E- G) Illustrative laser Doppler images and quantification of hindlimb blood flow recovery after femoral artery ligation in mice treated with FITC-traC compared to control animals. L: ligated, NL: non-ligated, T: tail. (n=5, E). Illustrative examples of the thigh (F) and calf (G) CD31 immunofluorescent staining (red) and its quantification, as well as Gapdh- normalized Cdh5 expression by real time PCR at day 7 post femoral artery ligation in mice treated with FITC-traC compared to control animals (n=5). Nuclei are stained blue with DAPI. Bar=200μm. *: P<0.05, **: P<0.01. Statistical significance was assessed by two-way ANOVA and Tukey’s multiple comparison test (B and D) or Student t-test (E, F and G).

Article Snippet: Subsequently, either 4 µl of FITC-traC (0.75 µg/µl), 4 µl of recombinant human VEGF (1 µg/µl, R&D Systems), or 4 µl of a solution containing both FITC-traC (0.75 µg/µl) and VEGF (1 µg/µl) was added and mixed.

Techniques: In Vivo, Staining, Ligation, Control, Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Regulation of angiogenesis by signal sequence-derived peptides

doi: 10.1101/2024.08.22.609269

Figure Lengend Snippet: FITC-traC enhances VEGF/VEGFR2 signaling in endothelial cells. (A) Illustrative immunofluorescence images showing FITC-traC (green) peptide penetration into the cells over time. Nuclei are stained blue with DAPI. Bar = 20μm. (B) Illustrative immunofluorescent images showing co-localization of FITC-traC (green) with VEGFR2 (red) on the cell membrane (arrowheads) 15 minutes after adding FITC-traC to endothelial cells. Nuclei are stained blue with DAPI. Bar = 20μm. (C) Western blot analysis of VEGF induced VEGFR2, ERK, and Akt phosphorylation with and without FITC-traC (n=7). (D) Western blot analysis of the effect of FITC-traC on VEGF induced VEGFR2, ERK, and Akt phosphorylation in endothelial cells following siRNA-mediated DCBLD2 downregulation (n=5). (E) Co-immunoprecipitation of FITC-traC with VEGFR2 but not PDGFRβ following the addition of FITC-traC to endothelial cell cultures. FITC immunoprecipitation is followed by Western blotting for VEGFR2, PDGFRβ, or FITC. (F) Effect of FITC-traC on protein tyrosine phosphatases PTP1B and TCPTP interaction with VEGFR2 in endothelial cells. VEGFR2 immunoprecipitation is followed by Western blotting for VEGFR2, PTP1B or TCPTP. PTP1B: Protein tyrosine phosphatase 1B; TCPTP: T cell protein tyrosine phosphatase. ns: not significant; *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison post hoc test (C) or by repeated measures one-way ANOVA and Holm-Šídák’s multiple comparisons post hoc test (D).

Article Snippet: Subsequently, either 4 µl of FITC-traC (0.75 µg/µl), 4 µl of recombinant human VEGF (1 µg/µl, R&D Systems), or 4 µl of a solution containing both FITC-traC (0.75 µg/µl) and VEGF (1 µg/µl) was added and mixed.

Techniques: Immunofluorescence, Staining, Membrane, Western Blot, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: Regulation of angiogenesis by signal sequence-derived peptides

doi: 10.1101/2024.08.22.609269

Figure Lengend Snippet: A poly leucine decapeptide mimics the effect of DCBLD2 SS traC on VEGF signaling. (A) Co-immunoprecipitation of the products of HA-tagged traC Part 1 or Part 2 constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2. (B) Co-immunoprecipitation of the products of HA-tagged traC Part 2 or its derivative constructs or a control HA-tagged GFP with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2. (C) Co-immunoprecipitation of the products of HA- tagged traC Part 2 L5VL5 sequence, or 9 or 16 poly-L-leucine constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2. (D) Co-immunoprecipitation of the products of HA- tagged polyleucine constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2. (E) Western blot analysis of VEGF induced VEGFR2 and ERK phosphorylation with and without FITC-L10 peptide (n=5). GAPDH was used as a loading control, with separate blots for total proteins (tp) and phosphorylated proteins (pp). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison post-hoc test.

Article Snippet: Subsequently, either 4 µl of FITC-traC (0.75 µg/µl), 4 µl of recombinant human VEGF (1 µg/µl, R&D Systems), or 4 µl of a solution containing both FITC-traC (0.75 µg/µl) and VEGF (1 µg/µl) was added and mixed.

Techniques: Immunoprecipitation, Construct, Cotransfection, Western Blot, Control, Sequencing, Comparison