期刊
ANALYTICAL BIOCHEMISTRY
卷 477, 期 -, 页码 13-20出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.02.020
关键词
Autophagy; Western blotting; Lysis buffer; LC3; p62
资金
- FPU predoctoral Fellowship (Ministerio de Educacion, Spain)
- Contrato Reincoporacion de Talentos (Gobierno de Extremadura, Spain)
- Ministerio de Economia y Competitividad, Spain [PI11/00040, PI14/00170]
- Ministerio de Economia y Competitividad, Spain
- CIBERNED [CB06/05/004, PI12/02280]
- Consejeria, Economia, Competitividad e Innovacion, Gobierno de Extremadura, Spain [GRU10054]
- Fondo Europeo de Desarrollo Regional (FEDER) from European Union
- Accion III postdoctoral contract (Universidad de Extremadura, Spain)
At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin Al (Baf. Al) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-l00, and sample buffer (SB) 1 x). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work. (C) 2015 Elsevier Inc. All rights reserved.
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