實驗室先前利用yeast two-hybrid的方法，發現TLE2 (transducin-like enhancer of split 2)會和Eps8有交互作用。TLE2 屬於一種transcription repressor，當和其他DNA-binding protein形成complex後，可以抑制target genes的轉錄作用。TLE proteins參與在眾多細胞的分化生成過程中，包括神經、骨骼、肌肉、及血液細胞。之前已有研究發現，當肌肉細胞分化的時候，Eps8的表現有下降的情形。因此我們推測Eps8會透過與TLE2之間的交互作用，而在肌肉細胞的分化過程中，扮演著抑制性的角色。首先，我們觀察到在v-Src transformed cells中，Eps8與TLE2有大量表達的情形。接著我們將細胞內容物分成細胞質與細胞核兩個部分，來觀察細胞內Eps8與TLE2的分布情形。我們發現 TLE2主要存在於細胞核中(75.5%)，而Eps8則是分於細胞質(56.4%)與細胞核內(43.6%)。之後，我們利用co-immunoprecipitation的方式，確認在細胞內，Eps8和TLE2之間會產生交互作用。更進一步發現此交互作用，主要是由p68 Eps8 isoform產生。之後，我們將p97Eps8及261-p97Eps8這兩種蛋白質的cDNA送入3T3細胞中，利用co-immunoprecipitation assay，觀察到只有261-p97Eps8會與TLE2有交互作用。當我們分析Eps8與TLE2在C2C12細胞中所扮演的角色，我們觀察到Eps8的表現量，隨著刺激的天數增加，肌細胞的分化有下降的情形；而TLE2的表現量則不受影響。我們將帶有Eps8與TLE2 cDNA的Tet-off vector送入C2C12細胞中，觀察到當細胞大量表現Eps8的時候，會抑制C2C12細胞分化產生myotube。因此，我們認為在細胞中大量表達Eps8，的確會抑制肌肉細胞的分化作用。

Eps8 (endothelial growth factor receptor substrate no.8) have two isoforms, p97Eps8 and p68Eps8. It can be phosphorylated by both EGFR and non-receptor tyrosine kinase (ex. Src). p97Eps8 overexpression will promote EGF-induced cell focus formation in cultured dish and tumor production in mice. However, down-regulation of p97Eps8 inhibits v-Src transformed cells growth and tumor formation. Utilizing yeast two-hybrid assay, TLE2 (transducin-like enhancer of split 2) was found to interact with Eps8. TLE family proteins have been shown to involve in many kinds of cell differentiation, including muscle cells. Researches revealed that TLE proteins can promote differentiation of myoblast into myotube. Since Eps8 expression was reduced when mouse myoblasts differentiate, we hypothesized that the interaction between Eps8 and TLE2 may play an important role in regulating muscle differentiation. To demonstrate this, first, we found in v-Src transformed cells, TLE2 as well as Eps8 is overexpressed. After fractionation of cell lysates into cytoplasm and nuclear proteins, we observed that, TLE2 was mainly present in nucleus, while Eps8 was localized in both cytosol and nucleus. Moreover, we immunoprecipitated Eps8 from IV5 cell lysates and observed that, p68Eps8, but not p97Eps8, interacts with TLE2. Then, we transfected to TLE2 with either p97Eps8 or 261- p97Eps8 into 3T3 cells. We found only 261-p97Eps8 can significantly interact with TLE2. Next, we examined whether Eps8 plays a role in muscle differentiation of C2C12 cells. Indeed, we observed that Eps8 expression but not TLE2 expression was reduced when myotubes are formed in C2C12 cells. Furthermore, we generated Eps8 and TLE2 overexpressing C2C12 cells. We found that Eps8 overexpression abolished muscle differentiation of C2C12 cells will not differentiate to myotubes. Therefore, we confirmed that decreased Eps8 expression is required for muscle cell differentiation.

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