碩士 / 國立臺灣大學 / 分子與細胞生物學研究所 / 95 / The 14-3-3 proteins form a family of highly conserved acidic proteins in all eukaryotic cells with a subunit mass of approximately 30 kDa. 14-3-3 proteins were capable of interacting with more than 200 different phosphorylated proteins. The binding partners are involved in almost every cellular process, like cell cycle control, apoptosis and signal transduction. The zebrafish 14-3-3 gene family consists of 11 distinct 14-3-3 genes. Previously, our laboratory had cloned 9 members of the zebrafish 14-3-3 gene family. In our study, we have expressed and purified GST fusion proteins and His-tagged fusion proteins of the 9 zebrafish 14-3-3 protein from E.coli. His-tagged 14-3-3 recombinant proteins were then injected into rabbit to obtain polyclonal antibodies against 14-3-3 β2, ζ1 ,ζ2 ,ε1 and η; these antibodies were also characterized. Proteins pull downed by GST fusion 14-3-3-ζ1 in the carp’s heart extracts were analyzed to identify potential interacting partners of 14-3-3-ζ1. 14 proteins, including β-actin-1, were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the 14-3-3-ζ1 GST pull downed proteins. The interaction between 14-3-3-ζ1 and β-actin-1 was further confirmed by GST pull down and co-immunoprecipitation in COS-1 cells. Our results indicate that β-actin-1 is a binding partner of 14-3-3-ζ1 both in vitro and COS-1 cells. Furthermore, the interaction between other 14-3-3 isoforms and β-actin-1 were also analyzed. We demonstrated that all 14-3-3 family proteins are able to bind β-actin-1in the GST pull down assay in vitro. However, only three 14-3-3 isoforms including ζ1, ζ2 and η have the ability to interact with β-actin-1 in COS-1 cells.
| Identifer | oai:union.ndltd.org:TW/095NTU05061010 |
| Date | January 2007 |
| Creators | Hsu-Ping Huang, 黃旭平 |
| Contributors | Fore-Lien Huang, 黃火鍊 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 45 |
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