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首页> 外文期刊>Brain research. Brain research protocols >Measurement of the synthesis, turnover, and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons.
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Measurement of the synthesis, turnover, and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons.

机译：测量培养的大鼠海马神经元中α-和β-类红血球及非类红血球光谱的合成，转换和组装。

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摘要

We describe a method that has allowed us to measure the synthesis, turnover and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. For these studies, rat hippocampal cultures containing 74.5-83.0% neurons were established. B-27 (Gibco) supplement has been used to obtain an excellent long-term viability (up to 5 weeks) of hippocampal neurons in culture. For the synthesis, turnover, and assembly experiments the neurons were labeled with [35S]methionine, and chased with 10-fold excess of cold methionine for the turnover experiments. The cells were then lysed and immunoprecipitated with alpha, beta-erythroid, alpha, and beta-nonerythroid spectrin antibodies. Immunoprecipitated [35S]methionine-labeled spectrins of hippocampal neurons grown in vitro produced bands in 5% polyacrylamide minigels strong enough to be detected by the high sensitivity screens of a phosphorimager to generate graphs from which the synthesis or half-lives of alpha, beta-erythroid, alpha, and beta-nonerythroid spectrins were calculated. This method can be used to study the role of calpain, caspase-3, and the ubiquitin-proteasome system on the synthesis and turnover of erythroid and nonerythroid spectrins in resting and depolarized rat hippocampal neurons in culture.
机译：我们描述了一种方法，该方法使我们能够测量培养的大鼠海马神经元中α-和β-类红血球及非类红血球光谱的合成，转化和组装。对于这些研究，建立了包含74.5-83.0％神经元的大鼠海马培养物。 B-27（Gibco）补充剂已被用于在培养物中获得优异的海马神经元长期生存能力（长达5周）。对于合成，周转和组装实验，将神经元标记为[35S]蛋氨酸，并用10倍过量的冷蛋氨酸追赶进行周转实验。然后裂解细胞，并用α，β-类红血球，α和β-非类红血球蛋白抗体进行免疫沉淀。免疫沉淀的体外生长的海马神经元的[35S]蛋氨酸标记光谱在5％聚丙烯酰胺微凝胶中产生了足够强的条带，足以被磷光成像仪的高灵敏度屏幕检测到，从而产生图，从中可以合成α，β-计算了类红细胞，α和β-非类红细胞的光谱。该方法可用于研究钙蛋白酶，caspase-3和泛素-蛋白酶体系统在培养的静止和去极化大鼠海马神经元中类红血球和非类红血球光谱的合成和转换中的作用。

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