eoe sub articles

EoE Sub Articles

1. Lentivirus production
NOTE: The production and manipulation of lentiviral vectors was carried out according to the National Institutes of Health (NIH) guidelines for research involving recombinant DNA. The plasmid encoding the wild-type and A4V mutant SOD1 tagged with enhanced YFP (SOD1WT-YFP and SOD1A4V-YFP) are used. Both gene fusion products were amplified using the PCR primer pair 5&#8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3&#8242; and 5&#8242;-TAGCGG CCGCTACTTGTACAGCTC...

protein aggregate formation assay: a method to detect and quantify protein aggregation in cultured cells upon induction by proteasome inhibitor

Source: Lee, H. et al. <a href="https://www.jove.com/v/56425">Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/21172/21172_Figure1.jpg" /> 
Figure 1. SOD1 WT and A4V stable line generation. (A) Schematic diagram of lentiviral and packaging vectors (Packing and Envelop plasmids) for wild type and mutant SOD1 A4V lentivirus generation. (B) Selected confocal images of three cells (HEK-293, U2OS, and SH-SY5Y) transduced with the lentivirus wild type SOD1 (WT) and mutant SOD1 (A4V). (C) Representative ima...

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
...

Removal of misfolded proteins in the cell is majorly regulated by the cellular ubiquitin-proteasome system, wherein the protein is first recognized and tagged by ubiquitin, a small regulatory protein, and trafficked to the proteasome, a multiprotein complex, for hydrolysis and clearance.
Impaired ubiquitin-proteosome system activity, a common occurrence in neurodegenerative diseases, can cause misfolded proteins to accumulate within the cells, forming insoluble protein aggregates with cytotoxic effects.
To visualize and quantify protein aggregation in vitro, obtain a suspension of transduced mammalian cells - expressing a fluorescent-labeled mutant protein that adopts a misfolded conformation with exposed hydrophobic residues - in suitable media.
Pipette increasing concentrations of a proteasome inhibitor to the wells of a black multi-well plate to minimize background fluorescence during imaging. Seed the mammalian cell suspension into the wells. Incubate to allow the cells to adhere to the plate bottom and proliferate.
Once inside, the proteasome inhibitor binds to the proteolytic sites of the proteasome and blocks its activity, causing the expressed mutant proteins to accumulate, which then interact via their exposed hydrophobic residues and form cytoplasmic aggregates, eventually leading to cell death.
Add a fluorescent DNA-binding dye to stain the cell nuclei. Image the plate. The fluorescent mutant protein aggregates appear dispersed within the unstained cytoplasm.
Wells containing higher proteasome inhibitor concentrations exhibit higher protein aggregate numbers to cell count ratio than wells with lower proteasome inhibitor concentrations.

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Overview