Lee, Hyunjung Ban, Eunmi Kim, Eunice EunKyeong Yoo, Young Sook Lee, Daekee Song, Eun Joo 2024-01-20T08:34:51Z 2024-01-20T08:34:51Z 2021-09-02 2014-10 0173-0835 https://pubs.kist.re.kr/handle/201004/126287 Protein degradation by the ubiquitin-proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS-dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)-fusion of the ubiquitin substrate ribophorin 1 (GFP-RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP-RPN1 and RFP in cell lysates were performed in an untreated capillary (75 mu m x 50 cm) and 100 mM Tris-CHES buffer (pH 9.0) containing 10 mM SDS. GFP-RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP-RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP-RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS-dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays. English WILEY IRREVERSIBLE INHIBITOR PROTEASOME SYSTEM 26S PROTEASOME PROTEIN EXPRESSION ALZHEIMERS PATHWAY LIGASE CELLS Quantitative analysis of a ubiquitin-dependent substrate using capillary electrophoresis with dual laser-induced fluorescence Article 10.1002/elps.201400308 1 ELECTROPHORESIS, v.35, no.20, pp.2978 - 2985 ELECTROPHORESIS 35 20 2978 2985 scie scopus 000343833000012 2-s2.0-84907876526 Biochemical Research Methods Chemistry, Analytical Biochemistry & Molecular Biology Chemistry Article IRREVERSIBLE INHIBITOR PROTEASOME SYSTEM 26S PROTEASOME PROTEIN EXPRESSION ALZHEIMERS PATHWAY LIGASE CELLS CE with dual LIF GFP-RPN1 RFP Ubiquitin-dependent substrate Ubiquitin-proteasome system