Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells
Published: April 30, 2024
Abstract
Source: Husain, M. Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection. J. Vis. Exp. (2022)
The video demonstrates a method to assess caspase-mediated protein cleavage in virus-infected cells. Post-viral infection, caspases get activated and degrade various host cellular proteins and viral nucleoproteins while specific caspase inhibitors prevent this degradation, as confirmed by western blotting.
Protocol
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 sam…
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The authors have nothing to disclose.
Materials
| A549 cells | ATCC | CRM-CCL-185 | Human, epithelial, lung |
| Ammonium chloride | Sigma-Aldrich | A9434 | |
| Caspase 3 Inhibitor | Sigma-Aldrich | 264156-M | Also known as 'InSolution Caspase-3 Inhibitor II – Calbiochem' |
| Complete, Mini Protease Inhibitor Cocktail | Roche | 11836153001 | |
| Goat anti-NP antibody | Gift from Richard Webby (St Jude Children's Research Hospital, Memphis, USA) to MH | ||
| MDCK cells | ATCC | CCL-34 | Dog, epithelial, kidney |
| MG132 | Sigma-Aldrich | M7449 | |
| Minimum Essential Medium (MEM) | ThermoFisher Scientific | 11095080 | Add L-glutamine, antibiotics or other supplements as required |
| Odyssey Fc imager with Image Studio Lite software 5.2 | LI-COR | Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software. | |
| Pierce BCA Protein Assay Kit | ThermoFisher Scientific | 23225 | |
| Protran Premium nitrocellulose membrane | Cytiva (Fomerly GE Healthcare) | 10600003 | |
| Rabbit anti-actin antibody | Abcam | ab8227 | |
| Rabbit anti-cortactin antibody | Cell Signaling | 3502 | |
| SeeBlue Pre-stained Protein Standard | ThermoFisher Scientific | LC5625 | |
| Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100 | Merck | ||
| Trypsin, TPCK-Treated | Sigma-Aldrich | 4370285 |
