Journal: Journal of the American Chemical Society

Article Title: Cell-Surface Glyco-Engineering by Exogenous Enzymatic Transfer Using a Bifunctional CMP-Neu5Ac Derivative

doi: 10.1021/jacs.7b05358

Figure Lengend Snippet: Display of 1 on Ext1−/− cells using ST6GAL1 modulates FGF2 binding and cell growth rate. (a) FGF2 binding by flow cytometry. (b) Representative Western blots of phospho-ERK1/2 and total ERK1/2 levels of Ext1−/− cells and Ext1−/− cells displaying 1 stimulated with FGF2. (c) Relative phospho-ERK1/2 levels with respect to total ERK1/2 levels 30 min after stimulation with FGF2. (d) Cell growth rate of Ext1−/− control cells and Ext1−/− cells displaying 1 for 48 h. *P < 0.01.

Article Snippet: Avidin-AlexaFluor-488 conjugate, propidium iodide (PI) and Hoescht 33342 were purchased from ThermoFisher Scientific; mouse monoclonal anti-biotin antibody conjugated to peroxidase (HRP) was from Jackson ImmunoResearch Laboratories; mouse monoclonal antibody (mAb) to β -actin conjugated to HRP, anti-FGF2 antibody, goat anti-rabbit IgG conjugated to HRP, and goat anti-rabbit IgG conjugated to AlexaFluor-488 were purchased from Abcam; recombinant human FGF2 basic was purchased from R&D Systems; p44/42 MAPK (ERK1/2, rabbit mAb) and phospho-p44/42 MAPK (phospho-ERK, rabbit mAb) were purchased from Cell Signaling Technology Inc.; Dulbecco’s phosphate-buffered saline (DPBS) with Ca 2+ /Mg 2+ , DPBS without Ca 2+ /Mg 2+ , and Dulbecco’s modified eagle’s medium (DMEM) high glucose were purchased from ATCC.

Techniques: Binding Assay, Flow Cytometry, Western Blot, 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: 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

Journal: Cell

Article Title: Dedicated epithelial recipient cells determine pigmentation patterns.

doi: 10.1016/j.cell.2007.07.024

Figure Lengend Snippet: Figure 6. Foxn1 Regulates Melanocyte Behavior via Fgf2 (A and B) Immunofluorescent staining of wild-type (A) and transgenic (B) skin at P4. Fgf2 is shown in red. (C) Measurement of protein secretion by ELISA. Graph shows Fgf2 levels in medium conditioned by wild-type (WT) or transgenic (Krt5- Foxn1) primary keratinocytes. Results are from four independent experiments. (D) Measurement of transcripts by real-time RT-PCR. y axis shows Fgf2 mRNA levels in keratinocytes infected with recombinant adenovi- ruses. x axis shows time after the start of infection. Results are from three independent experiments. (E) ChIP analysis of Foxn1-DNA complexes. Graph shows outcomes for two sites of the Fgf2 locus. Control assays using normal IgG precip- itated negligible quantities of sites 1 and 2 (precipitated DNA was below the threshold needed for quantitation). Results are from four independent experiments. (F) Schematic diagram of murine Fgf2. Sites assayed in E are indicated. In panels (D) and (E), Ad is the empty viral vector; Ad-Foxn1 produces full-length Foxn1 protein. (G and H) TTA analysis of melanocyte localization following Fgf2 neu- tralization. The epidermis and underlying tissue are shown from trans- genic littermates. Tyrosinase-positive cells are red. Animals were in- jected with vehicle (G) or antibodies to Fgf2 (H). Injections of normal IgG produced the same result as injections of vehicle.

Article Snippet: Staining reactions used either a goat polyclonal antibody to Foxn1 (WHN G-20; Santa Cruz Biotechnology), rabbit polyclonal antibody to Fgf2 (H-131; Santa Cruz Biotechnology), guinea pig polyclonal to Krt31 (hHa1; PROGEN Biotechnik), rabbit polyclonal to Tyrp1 (aPEP1, the gift of V. Hearing; Virador et al., 2001), biotinylated rat monoclonal antibody to Kit (2B8; PharMingen/ BD Biosciences), or the mouse monoclonal AE13 (the gift of T-T. Sun; Lynch et al., 1986).

Techniques: Staining, Transgenic Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Infection, Recombinant, Control, Quantitation Assay, Plasmid Preparation, Produced

Journal: Cell

Article Title: Dedicated epithelial recipient cells determine pigmentation patterns.

doi: 10.1016/j.cell.2007.07.024

Figure Lengend Snippet: Figure 7. Model of Foxn1 Function in Skin At the start of terminal differentiation, Foxn1 stimulates epithelial cells (squares) to secrete Fgf2 and other molecular signals. In a site-depen- dent manner, these signals promote up to two processes. One process is pigmentation, as melanocytes (hexagon) form pigmentary units with the Foxn1-positive cells. The second process is growth, as neigh- boring cells multiply, which expands or renews the tissue. Within the Foxn1-expressing cells, Foxn1 modulates the expression of differenti- ation markers and prevents the mitogenic signals from feeding back, enabling the cells to differentiate properly (indicated by keratin filaments).

Article Snippet: Staining reactions used either a goat polyclonal antibody to Foxn1 (WHN G-20; Santa Cruz Biotechnology), rabbit polyclonal antibody to Fgf2 (H-131; Santa Cruz Biotechnology), guinea pig polyclonal to Krt31 (hHa1; PROGEN Biotechnik), rabbit polyclonal to Tyrp1 (aPEP1, the gift of V. Hearing; Virador et al., 2001), biotinylated rat monoclonal antibody to Kit (2B8; PharMingen/ BD Biosciences), or the mouse monoclonal AE13 (the gift of T-T. Sun; Lynch et al., 1986).

Techniques: Expressing