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…

Divulgaciones

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

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Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells. J. Vis. Exp. (Pending Publication), e22216, doi: (2024).

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