LC3B and ubiquitin are measured directly using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. The kits contain highly specific primary antibodies, DyLight™-conjugated Secondary Antibodies and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.
Autophagy and the ubiquitin proteasome system constitute two major intracellular protein degradation pathways that are essential during cell starvation, differentiation, aging and death. LC3B participates in the process of mammalian autophagy, also characterized as a caspase-independent programmed cell death. Autophagy is the catabolic process of sequestering organelles and long-lived proteins in autophagic vesicles, which are eventually digested by lysosomal machinery. The ubiquitin proteasome system controls the stability of numerous proteins that regulate progression through the cell cycle and apoptosis such as cyclins, cyclin-dependent kinases, tumor suppressors and NFkB. Once these proteins are tagged with a single ubiquitin molecule, other ligases are signaled to attach additional ubiquitin. The result is a polyubiquitinated protein that is directed to the proteasome, a large protein complex, where it is degraded. This multiplex kit enables detection of markers, such as ubiquitin and LC3B, that signify formation of polyubiquitin structures and autophagic vesicles, respectively.
This kit was optimized using the Thermo Scientific ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module but can be used with other Cellomics BioApplications. Thus, automated plate-handling, focusing, cell image acquisition and data analysis are combined in one HCS system to assay for compounds that regulate the autophagy and ubiquitination process. In addition to HCS instruments, cells stained using reagents in this kit can be viewed and analyzed by fluorescence and confocal microscopes.
Figure 2. Staining of LC3B and ubiquitin in HepG2 cells treated with media (non-treated) or with the proteasome inhibitor MG132 (20 µM) for 16 hours. The enlarged image illustrates LC3B and ubiquitin in the same HepG2 cell. Cells were stained according to the kit protocol and imaged using the ArrayScan VTI HCS Reader.
Figure 3. Dose response curves for LC3B and poly-ubiquitin in HepG2 cells. The average intensities of LC3B and poly-ubiquitin in the cytoplasm are plotted against various concentrations of indicated drugs. HepG2 cells were incubated with various concentrations of MG132, ALLN, Tg or Tm for 20 hours.
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