The process of autophagy, or lysosome-mediated degradation of damaged proteins and organelles in the cytosol, is a vital cellular process that acts as a quality control mechanism for proteins and organelles. The misregulation of autophagy can lead to an imbalance of cellular homeostasis and the subsequent development of disease. Therefore, the study of autophagy is at the forefront of neuroscience and cancer research, among others. In order to measure autophagic flux, many assays use the autophagy marker protein LC3. LC3, the mammalian homolog of yeast ATG8, is a ubiquitin like protein that is associated with the autophagosome during the autophagic process. More directly related to LC3 is the process of selective autophagy, where receptors such as p62, NBR1 and NDP52 possess an LC3-interacting region (LIR) to directly bind LC3. Using a LC3 antibody is a reliable way to monitor autophagic activity in various experimental methods, including western blot, immunocytochemistry, and flow cytometry. The following articles describe the use of an LC3 antibody in each of these methods.
LC3B/MAP1LC3B Antibody [NB600-1384] - LC3 antibody was tested in 50uM Chloroquine treated HeLa cells with Dylight 488 (green). Nuclei and alpha-tubulin were counterstained with DAPI (blue) and Dylight 550 (red). An antibody concentration of 0.1 ug/ml was used. Image objective 40x.
First, Clarke et al used an LC3A antibody in flow cytometry to better understand autophagy activation in systemic lupus and its requirement for plasmablast development. Lupus is a moderately severe autoimmune disease caused by the over growth of autoreactive B cells in the immune system, and autophagy first came into play when genetic studies found alterations in the ATG5 gene in lupus patients. The LC3A antibody was used in flow cytometry of CD19+ B cells, CD4+ T cells and CD14+ monocytes. The results showed that the lupus cell samples had a higher level of active autophagy versus healthy controls.
Next, Morell et al also used an LC3A antibody to study the up-regulated expression of LAMP2 and autophagy during neuroendocrine (NE) differentiation of prostate cancer cells. To create a NE model of prostate cancer, prostate cancer cells were incubated in serum-free media for six days. Analysis of these cell cultures showed an up regulation of NE specific markers as well as the lysosomal membrane protein LAMP2. The LC3A antibody was used in western blot to detect autophagosome accumulation both on cells with and without the lysosomal proteases E64 and pepstatin A. This method was used to confirm that the results weren’t merely showing autophagosome turnover or induction of autophagy with impaired turnover ability. In the end, they concluded that autophagy plays a role in neuroendocrine differentiation of these prostate cells, given that inhibition prevents their development.
Furthermore, Rashid et al used an LC3B antibody to investigate the role of muscle LIM protein/CSRP3 in autophagy. Muscle LIM protein, or MLP, is a microtubule protein found in muscle tissues, and muscles heavily rely on autophagy for their survival. Using an LC3B antibody in both immunocytochemistry and immunoprecipitation, they were able to show that the binding of MLP to LC3 controls autophagy in C2C12 myoblasts, and lack of autophagy lead to increased apoptosis activity in these cells. The co-localization of LC3 and MLP in the immunocytochemistry images was analyzed via phase contrast microscopy, and photos were rendered using an inverted microscope.
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