泛素
化学
半胱氨酸蛋白酶
凋亡抑制因子
细胞凋亡
蛋白酶体
夏普
细胞生物学
分子生物学
生物化学
程序性细胞死亡
生物
基因
作者
Arcan Guven,Wen-Fang Wu,Saurabh Patil,Karthiga Devi Gokul,Poornima Tekumalla,Suresh K Sharma,Anne R. Diers,Stephane Gesta,Vivek K. Vishnudas,Rangaprasad Sarangarajan,Mark D. Kellogg,Niven R. Narain,Michael A. Kiebish
标识
DOI:10.1016/j.jpba.2019.05.005
摘要
Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.
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