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

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

1. 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.
2. 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....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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