Routine Western blot to check autophagic flux: cautions and recommendations.
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 A1 (Baf. A1) 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-100, and sample buffer (SB) 1×). 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.
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain.
Rubén
R
Gómez-Sánchez
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain.
Elisa
E
Pizarro-Estrella
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain.
Sokhna M S
SM
Yakhine-Diop
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain.
Mario
M
Rodríguez-Arribas
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain.
José M
JM
Bravo-San Pedro
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain. Electronic address: jfuentes@unex.es.
José M
JM
Fuentes
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Departamento de Bioquímica y Biología Molecular y Genética, Universidad de Extremadura, F. Enfermería y Terapia Ocupacional, 10003 Cáceres, Spain. Electronic address: rosapolo@unex.es.
Rosa A
RA
González-Polo
D000818:Animals; D001343:Autophagy; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D002460:Cell Line; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats
D015415:Biomarkers; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C118585:P62 protein, human; D016601:RNA-Binding Proteins
Autophagy; LC3; Lysis buffer; Western blotting; p62