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Rat hippocampal slices (300 μm) were prepared from male Sprague-Dawley rats (100 to 150 g) as described (8) and incubated (three slices per tube) at 35°C for 50 min before pharmacological treatments. At the end of the experiment, slices were sonicated in a solution of SDS (200 μl, 1% w/v) and sodium orthovanadate (1 mM) in water at 100°C, placed in a boiling water bath for 5 min, and stored at - 20°C until biochemical analysis.
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Neurons, prepared from 17-day-old rat embryos, were cultured in a monolayer in serum-free conditions (16). Thirty minutes before drug application, the medium was replaced with artificial cerebrospinal fluid (8). After treatment, the fluid was aspirated and the cells were solubilized in 1% (v/v) Triton X-100, deoxycholate (1 mg/ml), SDS (1 mg/ml), 158 mM NaCl, 10 mM tris-HCl (pH 7.2), 1 mM sodium orthovanadate, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, and pepstatin A, leupeptin, and trasylol (50 μg/ml each).
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2-terminal fragment of rat FAK [residues 1 to 377 (19)] expressed in Escherichia coli as a hexahistidine fusion protein. SL42 was raised against a peptide (Fig. 4A) coupled to keyhole limpet hemocyanin.
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+ 19 and pCMV2-C 19 contained inserts corresponding to rat FAK cDNA with (FAK+) or without (FAK) the insertion coding for the three amino acids Pro-Trp-Arg (19). COS-7 cells were transfected in the presence of N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP, Boehringer) according to the manufacturer's instructions. Cells were lysed 72 hours after transfection.
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C. Giaume and J. Glowinski are gratefully acknowledged for critical reading of the manuscript. F.B. was supported by the European Commission Human Capital and Mobility Programme (CT940705), J.C.S. by ECOS (Comité: Evaluation orientation de la Coopération Scientifique) and the European Commission International Scientific Cooperation (CT940038), V.deF, by INSERM, and M.L.B. by Association Française contre les Myopathies.
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