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Autophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins, mature ribosomes and even entire organelles. The best studied autophagic marker is the LC3B and it is believed that only the amount of the LC3B-II correlates with the amount of the autophagic membranes. Whether the LC3A processing, aside to LC3B, is a valuable endogenous 'autophagic flux' marker is far less clear. The specificity of rabbit polyclonal antibodies to the LC3A and the LC3B was tested against the commercial available human recombinant proteins LC3A and LC3B. In order to measure 'autophagic flux' in mouse liver, lung, kidney and heart we used: (1) a lysosomotropic reagent chloroquine, which inhibit the intra-lysosomal acidification or their fusion with autophagosome, (2) nutrient starvation as an autophagic stimulus and (3) ionizing radiation, which is known to destabilize lysosomes. According to the immunoblotting work the LC3A protein follows discrete patterns of LC3A-I and LC3A-II changes in liver, lung, kidney and heart tissues of mice, whereas the LC3B protein didn't follow the same pattern under stressor conditions. We conclude that the endogenous LC3A processing is a major marker of autophagy flux in mouse model. Fractionated samples (soluble vs. membrane fractions) should be used in immunoblotting to allow discrimination between the LC3-I soluble and the LC3-II membrane protein and kinetics. Further, when dealing with in vivo models it is necessary to check the specificity of the antibodies used against the LC3A and LC3B proteins as their expression and responsiveness is not overlapping.

Original publication

DOI

10.4161/auto.7.11.16664

Type

Journal article

Journal

Autophagy

Publication Date

11/2011

Volume

7

Pages

1371 - 1378

Keywords

Adaptor Proteins, Signal Transducing, Animals, Antibody Specificity, Apoptosis Regulatory Proteins, Autophagy, Beclin-1, Chloroquine, Gene Expression Regulation, Heat-Shock Proteins, Hepatocytes, Humans, Immunohistochemistry, Liver, Male, Mice, Mice, Inbred BALB C, Microtubule-Associated Proteins, Organ Specificity, Protein Processing, Post-Translational, RNA, Messenger, Radiation, Ionizing, Sequestosome-1 Protein