LC3B is one of the mammalian Atg8 homologs and widely used as an autophagosome marker. Immediately after synthesis, LC3 is processed by Atg4 and becomes LC3-I. Upon induction of autophagy, the C-terminal glycine of LC3-I is conjugated to phosphatidylethanolamine, resulting in formation of membrane-bound LC3-II. Most LC3-II is thought be present on autophagosome membrane. The autophagosome subsequently fuses with a lysosome, where inside materials, including LC3-II, are degraded. The expression level of LC3-II generally correlates with the number of autophagosome.
<Immunofluorescence microscopy analysis of mouse embryonic fibroblasts (MEFs)>
MEFs were cultured in regular DMEM supplemented with 10% FBS (left) or DMEM without amino acids (right) for 1 hr. After fixation with 4% paraformaldehyde for 10 min at room temperature, they were permeabilized with 50 µg/ml digitonin for 5 min. Cells were then subjected to immunofluorescence microscopy using #CAC-CTB-LC3-2-IC anti-LC3 antibody at 1:100 dilution. Scale bar, 20 µm.
< Immuno-electron microscopy analysis of mouse embryonic fibroblasts (MEFs)>
MEFs were cultured in DMEM without amino acids for 2 hr. After fixation with 4% paraformaldehyde for 2 hr at room temperature, they were permeabilized with liquid nitrogen. Immuno-electron microscopy analysis (pre-embedding method) of endogenous LC3 was performed using #CAC-CTB-LC3-2-IC anti-LC3 antibody at 1:10 dilution. The gold labeling was intensified by using a silver enhancement kit (HQ silver enhancement kit, Nanoprobes, NY). Scale bar, 500 nm.
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