This study investigates the role of caspase-3 in the degradation of viral nucleoproteins and host proteins in virus-infected mammalian cells. By utilizing a caspase-3 inhibitor, the research aims to elucidate the impact of caspase-3 activity on protein cleavage and viral propagation.
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 Celsius under a 5% carbon dioxide atmosphere. On the next day, remove the old culture medium from the cells and wash the cells in each well twice with 1 milliliter of serum-free MEM.
Then infect the cells by adding 400 microliters of influenza A virus inoculum and place the plate for incubation for one hour. After incubation, remove the virus inoculum and wash the cells with MEM. Add 1 milliliter of serum-free MEM to each well, and incubate the cells as shown in the previous step.
After 24 hours, harvest the cells by scraping them with a 1-milliliter syringe plunger, and transfer them to a 1.5-milliliter tube. Centrifuge the tube at 12,000 g for two minutes at room temperature, and collect the supernatant. Wash the cell pellet with 250 microliters of phosphate-buffered saline and centrifuge. Remove the supernatant and add 100 microliters of cell lysis buffer. Mix by vortex.
Heat the tube at 98 degrees Celsius for 10 minutes to completely lyse the cells. Then, resolve equal amounts of protein from uninfected and infected samples by standard SDS polyacrylamide gel electrophoresis. After electrophoresis, transfer the protein gel to a nitrocellulose or PVDF membrane and perform western blotting. Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.
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