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Sympathetic neurons were isolated from superior cervical ganglia of neonatal rats (21). Cells were plated in compartmentalized chambers (Tyler Research Products) as described (8) and grown in growth medium [Dulbecco's modified Eagle's medium containing fetal bovine serum (10%), 5 μM arabinosylcytosine (Ara-C), and NGF (200 ng/ml)]. Medium was replaced every 3 days. After 4 to 7 days, medium within the chamber containing cell bodies was replaced with medium lacking NGF. This procedure resulted in the death of those neurons that had not extended processes into adjacent compartments. Neurons grown in center-plated chambers were used 2 to 3 weeks after plating. Neurons grown in side-plated chambers required a longer time to project through two barriers and were used 3 to 4 weeks after plating. For P-CREB immunocytochemistry experiments, medium was changed to contain a low concentration of NGF (2 ng/ml) for 48 hours before stimulation with NGF. Neurons were treated with NGF (200 ng/ml) and then fixed with acetone: methanol (1:1) for 3 min. Fixed cells were rinsed with phosphate-buffered saline (PBS) and permeabilized with PBS containing Triton X-100 (0.1%). After blocking with PBS solution containing horse serum (3%) and bovine serum albumin (BSA, 3%) at room temperature for 2 hours, cells were incubated with anti-P-CREB (1:1000 dilution) in the above solution at 4°C for 18 hours. Immune complexes were detected with an avidin biotin detection system (Vector Laboratories).
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NGF was covalently coupled to 1 μm-diameter microspheres by means of a carbodiimide cross-linking method. Amine-modified FluoSpheres (2% solids; Molecular Probes) were washed three times with solution 1 [2(N-morpholino)ethanesulfonic acid (0.1 M, pH 6.0)] using centrifugation and gentle resuspension. The FluoSpheres were then resuspended in solution 1 containing NGF (100 μg/ml) to a final concentration of 1% microspheres. EDAC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; Molecular Probes], which was freshly dissolved in solution 1, was then added to this suspension, and the mixture was rotated at room temperature for 2 hours. The cross-linking reaction was quenched by the addition of glycine (0.1 M, pH 6.0). Beads were then washed four times with a high-salt buffer (10 mM sodium phosphate, 1.8 mM potassium phosphate, 1 M sodium chloride, and 2.6 mM potassium chloride, pH 4.0) for 30 min per wash. The beads were next incubated overnight in the high-salt buffer (pH 7.4), washed four times the next day with high-salt buffer (pH 10.0) to remove adsorbed NGF, and then resuspended in PBS (pH 7.4) at a final concentration of 0.25% solids. To ensure that all adsorbed NGF was removed from the FluoSpheres, we subjected a set of FluoSpheres (control beads) to a similar procedure, except EDAC was left out of the cross-linking step. The bioactivity of NGF-coupled FluoSpheres was found to depend on the concentration of NGF and EDAC used. At the concentration of NGF used, we found that 2 to 4 μM EDAC was optimal for obtaining bioactive NGF-coupled beads; we also found that 4 μl/ml of the bead solution was sufficient to cover all cell surfaces.
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6 cells per plate) treated with control medium, control beads (4 μl/ml), NGF (100 ng/ml), or NGF-coupled beads (4 μl/ml) (13), and phosphotyrosine protein immunoblotting was done as described (22). Similar results were obtained by immunoprecipitating TrkA from primary cultures of sympathetic neurons (14).
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675. The immune complexes were visualized with an avidin biotin detection system (Vector Laboratories).
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K-252a appeared to selectively block TrkA and not other protein kinases necessary for CREB phosphorylation because it completely blocked CREB phosphorylation induced by NGF but not that induced by epidermal growth factor or forskolin (14).
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We thank A. Ghosh, A. Kolodkin, D. Linden, and members of the Ginty laboratory for discussions and comments on this manuscript and R. Adler, D. Anderson, R. Mains, A. Pieper, and C. Tsui for helpful suggestions. We also thank D. Kaplan for providing anti-panTrk (203) and H. Ruan for anti-P-Trk. Supported by a Pew Scholars Award, a Klingenstein Award in Neuroscience, an Alfred Sloan Research Fellowship, and an American Cancer Society Junior Faculty Research Award (D.D.G.); a fellowship from Ministero della Sanità of Italy (A.R.); March of Dimes Birth Defects Foundation research grant 5-FY95-1114; and the Alzheimer's Association-Charles Evans Pilot Research Award.
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