7.2 LC3B

LC3B:
1
Autophagy is a catabolic process in which autophagolysosomes degrade most cytoplasmic contents.
During autophagy, three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications. After synthesis, the carboxy-terminal of LC3 is rapidly cleaved, producing the cytoplasmic LC3-I form. During autophagy, through a ubiquitin-like system involving Atg7 and Atg3, LC3-I is lipidated into LC3-II, thereby associating LC3 with autophagic vesicles.
The presence of LC3 in autophagosomes and its conversion to LC3-II (which migrates downward in electrophoresis) are markers of autophagy occurrence.
2
The formation and degradation of LC3-I/II is a dynamic process: transient LC3-II expression cannot reflect the extent of autophagy, and it is necessary to use tool drugs (such as chloroquine) in combination to analyze autophagic changes.
Autophagosomes undergo a process of formation, fusion, and degradation, known as "autophagic flux".
Therefore, changes in LC3-II expression at a single time point cannot reflect changes in autophagy. It is necessary to use drugs that block lysosomal degradation (such as chloroquine, bafilomycin A1, etc.) to determine changes in the degree of cellular autophagy under intervention.
Chloroquine inhibits the degradation of LC3-II by suppressing lysosomal function (e.g., increasing its pH value); bafilomycin A1 is a potent V-ATPase inhibitor that blocks lysosome-dependent degradation.
Under the premise of "blocking degradation", the change in autophagy is determined based on the accumulation of LC3-II over a period of time.
3
There are four subtypes of mammalian LC3 proteins (LC3A, LC3B, LC3B2, and LC3C), and their expression varies depending on the tissue or cell type. It is necessary to select an appropriate target antibody based on the cell line or tissue used. By using anti-LC3A/B antibodies to detect both LC3A and LC3B simultaneously, the success rate of the experiment can be significantly increased.
4
Ultrasonication
As a lipidated and membrane-associated protein, LC3 should be subjected to brief ultrasonication on ice after cell lysis to promote protein solubilization and dissociation from the membrane. Ultrasonication method: Perform 3 ultrasonic pulses at 35%-40% of the maximum power, with each pulse lasting 10 seconds and a 10-second pause between pulses. For different instruments, the maximum power can be adjusted to approximately 200W.
5
If No Cell Ultrasonicator Is Available
If a cell ultrasonicator is not available, a 1mL syringe (with a needle) can be used, and repeated aspiration can achieve an effect similar to ultrasonication. The molecular weight of LC3-I is approximately 16 kDa. Since LC3-II is conjugated with a PE group, its theoretical molecular weight is higher than that of LC3-I. However, the negatively charged PE alters the properties of LC3-II, making it more hydrophobic and resulting in a higher migration rate during electrophoresis. Therefore, after electrophoresis, LC3-II migrates to a position corresponding to a smaller molecular weight (approximately 14 kDa). For small-molecular-weight proteins such as LC3, it is necessary to use a 15% separating gel and a 0.22 μm membrane for protein transfer.