Routine Western blot to check autophagic flux: cautions and recommendations.

Analytical Biochemistry
Rubén Gómez-SánchezRosa A González-Polo

Abstract

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 relatio...Continue Reading

References

Nov 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·E J BowmanK Altendorf
Jun 21, 2007·The Journal of Biological Chemistry·Serhiy PankivTerje Johansen
Jul 6, 2007·Autophagy·Noboru Mizushima, Tamotsu Yoshimori
Feb 11, 2010·Cell·Noboru MizushimaBeth Levine
Nov 26, 2010·Autophagy·Hong-Min NiWen-Xing Ding
Dec 30, 2010·Autophagy·Terje Johansen, Trond Lamark
Apr 27, 2012·Nature Reviews. Cancer·Eileen White
Sep 12, 2012·Autophagy·Daniel J KlionskyBrian Zuckerbraun
Mar 28, 2013·DNA and Cell Biology·Xiaolong LinZuo Wang
Jan 17, 2014·Autophagy·Leonor Miller-FlemingDaniel J Klionsky

❮ Previous
Next ❯

Citations

Mar 5, 2016·The Journal of Physiological Sciences : JPS·Jeong-Sun JuJong-Moon Jeong
Apr 8, 2016·Data in Brief·Rubén Gómez-SánchezRosa A González-Polo
Jul 29, 2015·Bioconjugate Chemistry·Sarah A EngelberthMagnus Bergkvist
Mar 16, 2018·Journal of Virology·María Cecilia GimenezLaura Ruth Delgui
May 8, 2020·Oxidative Medicine and Cellular Longevity·Steffen HäseliChristiane Ott
Jul 28, 2018·ACS Chemical Neuroscience·Dafina M Angelova, David R Brown

❮ Previous
Next ❯

Related Concepts

Related Feeds

Autophagy & Model Organisms

Autophagy is a cellular process that allows degradation by the lysosome of cytoplasmic components such as proteins or organelles. Here is the latest research on autophagy & model organisms

Amygdala and Midbrain Dopamine

The midbrain dopamine system is widely studied for its involvement in emotional and motivational behavior. Some of these neurons receive information from the amygdala and project throughout the cortex. When the circuit and transmission of dopamine is disrupted symptoms may present. Here is the latest research on the amygdala and midbrain dopamine.