The function of ubiquitin in intralysosomal protein degradation.

HA-c-Cbl在HEK 293细胞中表达并免疫沉淀。免疫沉淀法检测胰岛素受体在HIRc细胞中的表达。然后将两种免疫沉淀物与E1、作为E2的UbcH 7、ATP及其再生系统、泛素或其突变体混合。这些混合物在pH 6.0、6.5和7.2下反应。免疫印迹法检测泛素化胰岛素受体。当反应在pH7.2下进行时，胰岛素受体被胰岛素受体中的多个赖氨酸残基单泛素化，而当反应在pH6.0下进行时，胰岛素受体通过泛素分子中的Lys-48进行多聚泛素化。因此，我计划接下来分析重组c-Cbl在中性pH和酸性pH之间的结构变化。接下来，我计划分析表皮生长因子受体和血小板衍生生长因子受体的泛素化谱。最近，我发现了LDH-A（一种胞质蛋白）在氧化应激下的单泛素化，并在溶酶体中被隔离和降解。现在，我会在溶酶体膜的胞质表面寻找泛素受体。

HA-c-Cbl was expressed in HEK293 cells and immunoprecipitated. Insulin receptor expressed in HIRc cells was immunoprecipitaed. Then Both immunoprecipitants were mixed with E1,UbcH7 as an E2,ATP and its regenerating system, ubiquitin or its mutants. These mixtures were reacted in pH6.0, 6.5 and 7.2. Ubiquitinated insulin receptor were detected by immunoblot. When the reaction was done at pH7.2, insulin receptor were monoubiquitinated with multiple lysine residues in insulin receptor, whereas when the reaction was carried out at pH6.0, the insulin receptor was subjected to polyubiqutination through Lys-48 in ubiquitin molecules. Therefore, I projected next to analyze the structural change of recombinant c-Cbl between neutral pH and acidic pH.Next, I was planed to analyze the ubiquitination profiles of epidermal growth factor receptor and platelet derived growth factor receptor. These projects would be under going.Recently, I identified the monoubiquitination of LDH-A, a cytosolic protein, under oxidative stress was sequestered into and degraded in lysosomes. Now, I would search the ubiquitin receptor on the cytosolic surface of lysosomal membranes.