Nuclear matrix association of multiple sequence-specific DNA binding activities related to SP-1, ATF, CCAAT, C/EBP, OCT-1, and AP-1.
Nuclear matrix association of multiple sequence-specific DNA binding activities related to SP-1, ATF, CCAAT, C/EBP, OCT-1, and AP-1.
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与 SP-1、ATF、CCAAT、C/EBP、OCT-1 和 AP-1 相关的多种序列特异性 DNA 结合活性的核基质关联。
DOI：
10.1021/bi00084a003
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Stein,GS
中科院分区：
文献类型：
--
作者：
vanWijnen,AJ;Bidwell,JP;Fey,EG;Penman,S;Lian,JB;Stein,JL;Stein,GS
材料和方法蛋白质制剂。如前所述，从融合的大鼠ROS 17/2.8骨肉瘤细胞和持续增殖的人HeLa S3宫颈癌细胞中分离出核基质蛋白(Bidwell等人，1993)。通过分离的核的0.42Mkc1提取获得的蛋白质，在这里被操作定义为非基质核因子，通过如前所述的标准程序(Dignam等人，1983)分离(van Wijnen等人，1991b；van den Ent等人，1993)。蛋白质定量采用Bradford分析(皮尔斯)。凝胶位移分析。图1概述了凝胶位移分析中用作探针的寡核苷酸。探针是通过使用T4多核苷酸激酶用32P标记来制备的，如前面所述(van Wijnen等，1992)。结合反应类似于所描述的(Bidwell等人，1993)，在20-Pl体积中使用10 ng/Pl聚(Di-DC)-聚(Di-DC)、15%甘油、75 mM Kc1、16 mM HEPES、pH 7.5、0.15 mM EDTA和10fmol的探针DNA。所有绑定混合料
MATERIALS AND METHODSProtein Preparations. Nuclear matrix proteins were isolated as described previously from confluent rat ROS 17/2.8 osteosarcoma cells and from continuously proliferating human HeLa S3 cervical carcinoma cells (Bidwell et al., 1993). Proteins obtained by 0.42 Mkc1 extraction of isolated nuclei, operationally defined here as nonmatrix nuclear factors, were isolated by standard procedures (Dignam et al., 1983) as described previously (van Wijnen et al., 1991b; van den Ent et al., 1993). Quantitation of proteins was performed by Bradford analysis (Pierce). Gel Shift Analysis. Oligonucleotides used as probes in gel shift assays are summarized in Figure 1. Probes were prepared as described previously (van Wijnen et al., 1992) by labeling with 32P using T4 polynucleotide kinase. Binding reactions were performed similarly as described (Bidwell et al., 1993) in a 20-pL volume with 10 ng/pL poly (dI-dC)-poly (dI-dC), 15% glycerol, 75 mM KC1, 16 mM Hepes, pH 7.5, 0.15 mM EDTA, and 10 fmol of probe DNA. All bindingmixtures