1. Assessing the cleavage or degradation of proteins in Influenza A virus, IAV-infected cells by caspases
- Seed 3 x 105 Madin-Darby Canine Kidney (MDCK) or A549 cells per well in a 12-well cell culture plate.
NOTE: If using MDCK cells, ensure that the antibody against the protein of interest recognizes its canine species.
- Seed the wells in pairs, i.e., a control pair-one well for the uninfected-mock sample and one for the infected-mock sample, a test pair-one well for uninfected-inhibitor A sample and one for the infected-inhibitor A sample. Increase the number of pairs with each additional inhibitor.
- Incubate the cells at 37 °C under a 5% CO2 atmosphere overnight.
- The next day, infect the cells with IAV (an H1N1 subtype or another subtype).
- For this, prepare the virus inoculum by diluting virus stock in 400 µL of serum-free minimum essential medium (MEM) (see Table of Materials) at a multiplicity of infection (MOI) of 0.5-3.0 plaque-forming units (pfu)/cell (a factor of the doubling of cell number when calculating the MOI).
NOTE: For infecting MDCK cells, supplement the virus inoculum with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin at a final concentration of 1 µg/mL.
- Remove the old culture medium from the cells (step 1.3) and wash the cells with 1 mL/well of serum-free MEM 2x.
- Add 400 µL of virus inoculum (step 1.4.1) to the cells and incubate them for 1 h at 35 °C under a 5% carbon dioxide (CO2) atmosphere.
- In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., ammonium chloride, NH4Cl), or a proteasome inhibitor (e.g., MG132) (see Table of Materials) at a final concentration of 40 µM, 20 mM, or 10 µM, respectively, in serum-free MEM (see Table of Materials). The latter two inhibitors serve as a control. Dilute an equal volume of the solvent (if other than water) used to reconstitute one of the inhibitors in serum-free medium as a mock.
- Remove the virus inoculum and wash the cells as in step 1.5 (1x).
- Add the serum-free MEM, 1 mL/well, mock or supplemented with an inhibitor, to the cells and incubate the cells as in step 1.6. This time point is considered as 0 h infection.
- After 24 h, harvest the cells by scraping them with a 1 mL syringe plunger (rubber side) and transfer them into a 1.5 mL polypropylene tube.
- Centrifuge the tube at 12,000 x g for 1-2 min at room temperature. Collect the supernatant using a pipette.
NOTE: This supernatant could be discarded or used for a plaque assay to measure the titer of the released virus progeny.
- Wash the cell pellet with 250 µL of phosphate-buffered saline (PBS) by re-centrifugation as in step 1.11.
- Remove the supernatant and lyse the cells by adding 80-100 µL of cell lysis buffer (50 mM Tris (hydroxymethyl) aminomethane (THAM) hydrochloride (Tris-HCl), pH 7.4, 150 mM sodium chloride (NaCl), 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail, see Table of Materials) and vortexing.
- Heat the tube at 98 °C for 10 min to completely lyse and prepare the total cell lysate. Store the lysates at 4 °C for performing steps 1.15-1.17 the next day, but, for best results, finish these steps on the same day.
- Estimate the protein amount in each sample using a bicinchoninic acid (BCA) tableassay kit (see Table of Materials).
- Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE) along with molecular weight markers.
- Transfer the protein to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane (see Table of Materials).
NOTE: PVDF membrane may give a high background in some western blot imagers; check for compatibility.
- Perform western blotting to detect the protein of interest using the method described elsewhere.
- Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.
NOTE: If the protein level has recovered in the caspase inhibitor-treated infected sample lane, then the protein is cleaved or degraded by caspases. Otherwise, it is degraded by either lysosome or proteasome.
- Quantify the protein recovery by measuring its band intensity in each lane from at least three replicates of the same experiment and normalizing it with the corresponding loading control band.
- Use any contemporary imager and associated software (see Table of Materials) to image the western blots and quantify the protein recovery.
