Shared Decision Making and Communication.Scientific Discovery and the Future of Medicine.Health Care Economics, Insurance, Payment.Clinical Implications of Basic Neuroscience.Challenges in Clinical Electrocardiography.Values are the average (mean ± SEM) from 4 independent cultures, triplicate wells for each cpm indicates counts per minute. Asterisk indicates P<.05, and dagger, P<.01, with respect to untreated control double dagger, P<.01 compared with FGF-1 alone (10 ng/mL). Black bars indicate chlorate treatment alone stippled bars, heparinase and chlorate treatments. Combined heparinase and chlorate treatment abolished FGF-1–induced DNA synthesis, although this effect could be efficiently rescued by coaddition of low but not high doses of heparin. B, When DNA synthesis was measured instead of cell number increase, FGF-1 still stimulated DNA replication after chlorate treatment, presumably acting through preexisting heparan sulfate proteoglycans. Asterisk indicates P<.05, and dagger, P<.01, with respect to untreated control double dagger, P<.01 with respect to chlorate-treated controls section mark, P<.05 with respect to low-dose heparin treatment. Black bars indicate cell density at start of experiment gray bars, cell density after 3 days in culture. However, this effect was restored in the presence of low or high doses of heparin, the latter being significantly less elevated than the former. After sodium chlorate (NaClO 3) block of sulfation, Y79 cells were no longer able to respond mitotically to FGF-1. Addition of low or high doses of heparin (shown in the second row under the x-axis) did not greatly alter the inductive effect when FGF-1 was used at this dose. A, As before, addition of FGF-1 (10 ng/mL, depicted in first row below the x-axis) led to increased Y79 proliferation after 3 days in vitro compared with growth in unstimulated controls. Suppression of fibroblast growth factor 1 (FGF-1)–induced Y79 proliferation by perturbation of heparan sulfate proteoglycan coreceptors. Data are expressed as relative levels of cell numbers after 3 days in vitro compared with the original density (mean ± SEM), calculated from 3 independent cultures, quadruplicate wells for each. Addition of nonimmune IgG (10 μg/mL) did not inhibit Y79 growth (last column). B, Addition of FGF-1, 10 ng/mL, led to significant increases in cell numbers after 3 days in vitro, which were unchanged in the presence of blocking antibody MAB125 at 1 μg/mL, but completely prevented at 10 μg/mL. Studies were performed 3 times, with identical results. The use of FGF-1 alone led to intense staining of an 80-kDa band for FGFR1, whereas there was a reduction in intensity of immunostaining at 1 μg/mL and complete extinction of the signal at an antibody concentration of 10 μg/mL. A, Y79 cells were incubated with FGF-1 alone (100 ng/mL for 5 minutes) or in combination with the neutralizing antibody MAB125 (1 and 10 μg/mL), followed by lysis and immunoprecipitation with anti-FGFR1 and immunoblotting with antiphosphotyrosine. See the “Western Blotting” subsection of the “Methods” section for additional details.īlocking of fibroblast growth factor receptor 1 (FGFR1) phosphorylation and fibroblast growth factor 1 (FGF-1)–induced Y79 proliferation by anti-FGFR1 neutralizing antibody. Experiments were performed at least 3 times for each FGFR representative blots are shown. Stimulation is designated as follows: h, heparin (10 ng/mL) alone F, FGF-1 (100 ng/mL) alone Fh, FGF-1 (100 ng/mL) plus heparin (10 ng/mL) and FH, FGF-1 (100 ng/mL) plus heparin (10 μg/mL). B, Y79 retinoblastoma cells were stimulated for 5 minutes with FGF-1 and different concentrations of heparin, then immunoprecipitated with anti-FGFR1 and probed with antiphosphotyrosine. A, Immunoprecipitation using anti-FGFR1 and subsequent phosphotyrosine immunoblotting disclosed a major band of approximately 80 kDa (arrow at right), which varied in intensity according to the duration of stimulation (shown in minutes at the top of the bar) with FGF-1, 100 ng/mL. In the presence of phosphatase inhibitors, there was no background tyrosine phosphorylation under nonstimulated conditions (0 at bottom of figure). Fibroblast growth factor 1 (FGF-1)–induced tyrosine phosphorylation of FGF receptor 1 (FGFR1) in Y79 cells demonstrated by Western blotting of Y79 lysates with antiphosphotyrosine antibody of proteins immunoprecipitated with anti-FGFR1 under either nonstimulated or FGF-1–stimulated conditions.
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