1. The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.
- Author
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Wang W, Chen Z, Billiar TR, Stang MT, and Gao W
- Subjects
- Amino Acid Sequence, Animals, Autophagy-Related Proteins, Blotting, Western, Cell Line, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Electrophoresis, Polyacrylamide Gel, Humans, Leucine genetics, Leucine metabolism, Lipids chemistry, Lysine genetics, Lysine metabolism, Mice, Microtubule-Associated Proteins genetics, Molecular Sequence Data, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Precursors genetics, Protein Structure, Tertiary, Rats, Species Specificity, Autophagy genetics, Microtubule-Associated Proteins metabolism, Protein Precursors metabolism
- Abstract
LC3 is widely used marker for macroautophagy assays. After translation pro-LC3 is processed by Atg4 to expose C-terminal glycine residue for downstream conjugation reactions to accomplish the conversion of LC3-I to LC3-II. SDS-PAGE based Western blot (Wb) is generally utilized to quantify LC3-II levels where the LC3-I band migrates slower than LC3-II. We found that pro-human LC3B migrated at similar rate as LC3B-II in SDS-PAGE. The carboxyl-terminal five amino acids, particularly Lysine(122) and Leucine(123) of human LC3B play a major role in the faster migration of unprocessed LC3B, rendering it indistinguishable from LC3B-II in Wb assays. The unique faster migration of unprocessed LC3B than LC3B-I is also revealed in mouse LC3B, rat LC3B and rat LC3 but not in human LC3C. Our findings for the first time define pro-LC3 migration patterns for LC3 family member from human, mouse and rat species in SDS-PAGE. These findings provide a reference for pro-LC3 band patterns when Atg4 function is inhibited.
- Published
- 2013
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