Signaling between the extracellular matrix and the cytoskeleton: tyrosine phosphorylation and focal adhesion assembly.
Signaling between the extracellular matrix and the cytoskeleton: tyrosine phosphorylation and focal adhesion assembly.
复制标题
细胞外基质和细胞骨架之间的信号传导：酪氨酸磷酸化和粘着斑组装。
DOI：
10.1101/sqb.1992.057.01.024
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Turner,CE
中科院分区：
文献类型：
--
作者：
Romer,LH;Burridge,K;Turner,CE
方法细胞培养.大鼠胚胎成纤维细胞（REF 52）在补充有10%胎牛血清、50单位/ml青霉素和50 μ g/ml链霉素的Dulbecco改良Eagle培养基中生长。小鼠NIH-3 T3细胞在含有10%小牛血清的相同培养基中生长。细胞在使用前传代1-3天。对于粘附实验，将NIH-3 T3细胞与放线菌酮以25 μ g/ml孵育2小时。将细胞胰蛋白酶化，在含有1 mg/ml火鸡蛋白色胰蛋白酶抑制剂（Sigma Chemical Co.，St. Louis，密苏里州），再次在无血清培养基中洗涤，并在含有25 μ g/ml放线菌酮的无血清培养基中铺板。REF 52细胞不用放线菌酮处理。将这些细胞胰蛋白酶化，在含血清培养基中洗涤一次，在无血清培养基中洗涤两次，然后在无血清培养基中铺板。用人血浆纤连蛋白包被培养皿或盖玻片（纽约血液中心）以0.1 mg/ml在37 ℃下90分钟或在4 ℃下过夜。培养皿用0.5 mg/ml的聚-L-赖氨酸（Sigma）在37 ℃下包被90分钟。（Gibco BRL，盖瑟斯堡，马里兰州），将细胞与溶解在DMSO中的875 nM除草霉素A预孵育，并在24小时后接种。将这些细胞接种在纤连蛋白包被的表面上。
METHODSCell culture. Rat embryo fibroblasts (REF52) were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 pg/ml streptomycin. Mouse NIH-3T3 cells were grown in the same medium with 10% calf serum. Cells were passaged 1-3 days prior to use. For adhesion experiments, the NIH-3T3 ceils were incubated with cycloheximide at 25/~ g/ml for 2 hours. The cells were trypsinized, washed in serum-free medium containing 1 mg/ml turkey egg white trypsin inhibitor (Sigma Chemical Co., St. Louis, Missouri), washed again in serum-free medium, and plated in serum-free medium containing 25 p~ g/ml cycloheximide. REF52 cells were not treated with cycloheximide. These cells were trypsinized, washed once in medium containing serum, and washed twice in serum-free medium before plating in serum-free medium. Culture dishes or coverslips were coated with human plasma fibronectin (New York Blood Center) at 0.1 mg/ml either for 90 minutes at 37~ or overnight at 4~ Culture dishes were coated with poly-L-lysine (Sigma) at 0.5 mg/ml for 90 minutes at 37~ In experiments using herbimycin A (Gibco BRL, Gaithersburg, Maryland), cells were preincubated with, and plated 24 hours later in the presence of, 875 nM herbimycin A dissolved in DMSO. These cells were plated on fibronectin-coated surfaces.