Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells

Begin with virus-infected mammalian cells that produce multiple caspases, including caspase-3.

Add a growth medium to the control well and a caspase-3 inhibitor-supplemented medium to the test well.

In the control well, caspase-3 recognizes and degrades the viral nucleoproteins and host proteins essential for viral propagation.

In the test well, the inhibitor enters the cell, inhibiting caspase-3 activity and preventing protein degradation.

Dislodge cells from the plate. Add a lysing reagent and heat to induce cell lysis, releasing intracellular proteins.

Load both samples on an SDS-polyacrylamide gel and electrophorese to separate proteins based on size.

After electrophoresis, transfer the gel to a blotting membrane.

Treat the membrane with primary antibodies against viral nucleoproteins and host proteins.

Wash and overlay with fluorophore-conjugated secondary antibodies binding with primary antibodies, generating fluorescent bands.

The higher fluorescence in the test sample indicates reduced caspase-3 mediated viral and host protein cleavage compared to the control.

To begin, seed MDCK or A549 cells in a 12-well cell culture plate, incubate the cells overnight at 37 degrees Celsi