Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C.
Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C.
复制标题
人胰岛素受体的苏氨酸 1336 是蛋白激酶 C 磷酸化的主要靶点。
DOI：
10.1021/bi00459a020
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Czech,MP
中科院分区：
文献类型：
--
作者：
Lewis,RE;Cao,L;Perregaux,D;Czech,MP
方法细胞培养：3T3/HIR成纤维细胞（Whittaker etal ., 1987）保存在含有10%胎牛血清的Dulbecco改良Eagle培养基中。胰岛素受体的体内磷酸化和免疫沉淀。细胞在37℃无磷酸盐Dulbecco改良Eagle培养基中孵育1小时，加入[32P]正磷酸盐至终浓度为2 mCi/mL。再继续孵育2小时。细胞在37℃下加或不加100 nM PMA孵育20分钟，培养物置于冰上，溶解于10 mM Hepes， pH 7.8，含1% Triton X-100, 500 mM NaCl, 5 mM EGTA， 30 mM焦磷酸钠，50 mM氟化钠，100 mM正钒酸钠，0.1%牛血清白蛋白，10 Mg/mL胰肽和0.1 mM苯甲磺酰氟（bufferA）。不溶性物质在离心液中以15000 rpm离心15分钟去除。将细胞上清加入到aIR-1中，aIR-1是一种针对胰岛素受体的单克隆抗体（Kull等人，1983），该抗体与蛋白a - se -预结合
MethodsCell Culture. 3T3/HIR fibroblasts (Whittaker et al., 1987) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. In Vivo3 Phosphorylation and Immunoprecipitation of In-sulin Receptors. Cells were incubated for 1 h in phosphate-free Dulbecco’s modified Eagle’s medium at 37 C, at which time [32P] orthophosphate was added to a final concentration of 2 mCi/mL. Incubation was continued for an additional 2 h. Cells were then incubatedwith or without 100 nM PMA for 20 min at 37 C, and the cultures were placed on ice and solubilized in 10 mM Hepes, pH 7.8, containing 1% Triton X-100, 500 mM NaCl, 5 mM EGTA, 30 mM sodium pyro-phosphate, 50 mM sodium fluoride, 100 mM sodium ortho-vanadate, 0.1% bovine serum albumin, 10 Mg/mL leupeptin, and 0.1 mM phenylmethanesulfonyl fluoride (bufferA). In-soluble matter was removed by centrifugation for 15 min at 15 000 rpm in a microfuge. Cell supernatants were added to aIR-1, a monoclonal antibody against the insulin receptor (Kull et al., 1983), which was prebound to protein A-Se-