Binding of Insulin Receptor Substrate 1 PH Domain to Protein Kinase C
To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total...
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Published in | Sheng wu hua hsüeh yü sheng wu wu li hsüeh pao Vol. 31; no. 4; p. 400 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
China
1999
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Abstract | To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total RNAs were isolated and purified from fresh liver and mRNAs were reversely transcribed into cDNAs. After PCR the fragments of the DNA were cloned into vector pUC19. The sequence of the fusion gene was confirmed by sequencing, and the gene was correctly expressed in E.coli as fusion protein with glutathione S- transferase(GST). The fusion protein was purified by glutathione agarose beads, then was incubated with the lysate of Jurkat cells. After SDS-PAGE, the proteins were transferred to PVDF membrane, and an anti-PKC antibody was used to detect binding between the PH domain with PKC. The sequence of the gene encoding for the PH domains was confirmed to be correct, and the PH domain was successfully expressed in E.coli JM 109 in soluble form. Western blots confirmed the binding of the PH domain with PKC in vitro. In conclusion, purified IRS-1 PH domain GST fusion protein was obtained and its biological activity was confirmed. |
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AbstractList | To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total RNAs were isolated and purified from fresh liver and mRNAs were reversely transcribed into cDNAs. After PCR the fragments of the DNA were cloned into vector pUC19. The sequence of the fusion gene was confirmed by sequencing, and the gene was correctly expressed in E.coli as fusion protein with glutathione S- transferase(GST). The fusion protein was purified by glutathione agarose beads, then was incubated with the lysate of Jurkat cells. After SDS-PAGE, the proteins were transferred to PVDF membrane, and an anti-PKC antibody was used to detect binding between the PH domain with PKC. The sequence of the gene encoding for the PH domains was confirmed to be correct, and the PH domain was successfully expressed in E.coli JM 109 in soluble form. Western blots confirmed the binding of the PH domain with PKC in vitro. In conclusion, purified IRS-1 PH domain GST fusion protein was obtained and its biological activity was confirmed. |
Author | Wang, Ji-Cun Ji, Shao-Ping Su, Cheng-Zhi Liu, Xin-Ping Yao, Li-Bo Bai, Yu-Jie |
Author_xml | – sequence: 1 givenname: Shao-Ping surname: Ji fullname: Ji, Shao-Ping email: jishaoping@hotmail.com organization: Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China. jishaoping@hotmail.com – sequence: 2 givenname: Li-Bo surname: Yao fullname: Yao, Li-Bo – sequence: 3 givenname: Yu-Jie surname: Bai fullname: Bai, Yu-Jie – sequence: 4 givenname: Ji-Cun surname: Wang fullname: Wang, Ji-Cun – sequence: 5 givenname: Xin-Ping surname: Liu fullname: Liu, Xin-Ping – sequence: 6 givenname: Cheng-Zhi surname: Su fullname: Su, Cheng-Zhi |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/12114993$$D View this record in MEDLINE/PubMed |
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Title | Binding of Insulin Receptor Substrate 1 PH Domain to Protein Kinase C |
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