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Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: Endothelial cells (BME-1) were cultured on Matrigel®-coated surfaces and incubated either without (A, C and E) or with the addition of FGF2 and VEGF-165 (B, D and F) for 3 hrs. Panels A and B show phase contrast images, panels C and D show computer-generated projections of panels A and B used for quantification and panels E and F show the superimposed images. Panel G shows quantification of tube length (mean ± SEM of triplicate determinations in each case) as indicated. *** p < .001 relative to cells receiving no growth factors (left four bars) or to cells receiving growth factors without HIP/RPL29 (right three bars).

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Cell Culture, Incubation, Generated

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: Mouse aortic outgrowth assays were performed and quantified by computer-based morphometric analyses as described in Materials and Methods. Panels A–F show 8 day outgrowths from cultures grown in the presence of EBM with the following additions: A, none (control); B. 40 ng/ml FGF2; C, 40 ng/ml VEGF-165; D, 40 μg/ml HIP/RPL29; E, 40 ng/ml FGF2 plus 40 μg/ml HIP/RPL29; F, 40 ng/ml VEGF-165 plus 40 μg/ml HIP/RPL29. Panel G shows the quantitation of results of these types of assays and demonstrates near complete inhibition of outgrowth in the presence of HIP/RPL29 in all cases. * p < 0.001 vs. corresponding growth factor treatment in the absence of HIP/RPL29.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Control, Quantitation Assay, Inhibition

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: FGF2 (A) or VEGF-165 (B) were preincubated with perlecan domain I in a solid phase assay, unbound growth factor rinsed off and the substrates subsequently incubated with the indicated concentrations of HIP/RPL29 (HIP; filled circles) or lysozyme (LYS; open circles) for 2 hr. The surface was rinsed again and bound growth factor determined by ELISA as described in Materials and Methods. The points indicate the means ± SEM of triplicate determinations from a representative experiment.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: FGF2 was bound to a solid phase perlecan domain I substrate as described in Materials and Methods and Figure 5. In panel A, complexes subsequently were incubated with HPSE at the indicated concentrations for 24 hr, rinsed to remove unbound/released FGF2 and bound FGF2 measured by ELISA as described in Materials and Methods. The negative control was the perlecan domain I substrate not exposed to FGF2. In panel B, 5 μg/ml HPSE was incubated in the presence of the FGF2 bound to perlecan domain I for 24 hr at pH 5 or pH 7.2 as indicated and FGF2 release assayed by ELISA as described in Materials and Methods. FGF2 that remained bound at either pH 5.0 or 7.2 in the absence of HPSE served as a buffer only control. The bars represent the means ± SEM of triplicate determinations in each case.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Negative Control, Control

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: FGF2 was bound to a solid phase perlecan domain I substrate as described in the legend to Figure 5. This complex subsequently was incubated with HPSE (5 μg/ml) alone or in the presence of 0.5, 5 or 40 μg/ml HIP/RPL29 as indicated on the figure. After 24 hr, the surfaces were rinsed to remove unbound/released FGF2 and bound FGF2 measured by ELISA as described in Materials and Methods. The negative control was the perlecan domain I substrate not exposed to FGF2. The bars represent the means ± SEM of triplicate determinations from a representative experiment in each case.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Negative Control