Direct proteasome inhibition by clasto-lactacystin β-lactone permits the detection of ubiquitinated p21waf1 in ML-1 cells

被引：19

作者：

Fukuchi, K ^{[1
]}

Maruyama, H ^{[1
]}

Takagi, Y ^{[1
]}

Gomi, K ^{[1
]}

机构：

[1] Showa Univ, Sch Med, Dept Clin Pathol, Shinagawa Ku, Tokyo 1428666, Japan

来源：

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH | 1999年 / 1451卷 / 01期

关键词：

ubiquitin; proteasome; p21(waf1); beta-lactone; DNA damage;

D O I：

10.1016/S0167-4889(99)00081-6

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1 x 10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination. (C) 1999 Elsevier Science B.V. All rights reserved.

引用

页码：206 / 210

页数：5

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