Direct proteasome inhibition by clasto-lactacystin beta-lactone permits the detection of ubiquitinated p21(waf1) in ML-1 cells.
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 1x10(-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.
Fukuchi
K
K
Department of Clinical Pathology, Showa University, School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan. kfukuchi@med.showa-u.ac.jp
Maruyama
H
H
Takagi
Y
Y
Gomi
K
K
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007783:Lactones / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D007783:Lactones; D009097:Multienzyme Complexes; D014452:Ubiquitins; C067713:lactacystin; C098869:clasto-lactacystin beta-lactone; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D003676:Deferoxamine; D000111:Acetylcysteine