Retinoic acid receptor gamma: specific immunodetection and phosphorylation
Retinoic acid receptor gamma: specific immunodetection and phosphorylation
复制标题
视黄酸受体γ：特异性免疫检测和磷酸化
作者：
C. Rochette;Y. Lutz;M. Saunders;I. Scheuer;M. Gaub;P. Chambon
合成与人类和小鼠视黄酸受体γ 1（分别为hRAR- γ 1和mrar - γ 1）特有的cdna推导氨基酸序列相对应的肽段，用于产生抗rar - γ 1抗体。选择了4个单抗，分别针对A1区（Ab1 γ (A1)）、F区(Ab2 γ （mF)和Ab4 γ (hF)）和D2区（Ab5 γ (D2)）中发现的肽。这些抗体在转染含有rar - γ -1 cdna的表达载体的COS-1细胞中特异性免疫沉淀并被免疫印迹法识别。它们都能与人和小鼠的rar - γ - 1蛋白反应，除了Ab4 γ (hF)是har - γ - 1特异性的。兔多克隆抗体，直接针对mRAR-gamma 1 F区域的肽（RP gamma (mF)），发现对小鼠RAR-gamma 1蛋白具有特异性。此外，在凝胶阻滞/移位实验中，抗体特异性地阻滞了RA反应元件（RARE）获得的复合物的迁移。针对D2区和F区提出的抗体也识别出RAR-gamma 2亚型，该亚型仅在A区与RAR-gamma 1不同。另一方面，针对RAR-gamma 1 A1区的抗体（Ab1 gamma (A1)）仅与RAR-gamma 1蛋白反应。这些抗体使我们能够在小鼠胚胎和F9胚胎癌细胞核提取物中检测mrar - γ - 1和γ - 2亚型的存在。他们也被用来证明mrar - γ - 1蛋白可以被磷酸化，并且磷酸化主要发生在nh2末端的A/B区。
Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR- gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR- gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR- gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.
登录
查看更多内容
DOI：
10.1016/s0021-9258(20)89482-4
发表时间：
1991-04
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. Bodwell;E. Ortí;J. Coull;D. Pappin;L. I. Smith;F. Swift
通讯作者：
J. Bodwell;E. Ortí;J. Coull;D. Pappin;L. I. Smith;F. Swift
影响因子：
11.4
作者：
ZELENT, A;MENDELSOHN, C;CHAMBON, P
通讯作者：
CHAMBON, P
鸡孕酮受体磷酸化位点的激素调节和鉴定。
DOI：
--
发表时间：
1990
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Denner,LA;Schrader,WT;O'Malley,BW;Weigel,NL
通讯作者：
Weigel,NL
DOI：
10.1002/j.1460-2075.1991.tb08055.x
发表时间：
1991-05
期刊：
The EMBO Journal
影响因子：
--
作者：
G. Vasios;S. Mader;J. D. Gold;M. Leid;Y. Lutz;M. Gaub;P. Chambon;L. Gudas
通讯作者：
G. Vasios;S. Mader;J. D. Gold;M. Leid;Y. Lutz;M. Gaub;P. Chambon;L. Gudas
影响因子：
56.9
作者：
DENNER, LA;WEIGEL, NL;OMALLEY, BW
通讯作者：
OMALLEY, BW