Determination of Protein Ubiquitination After Protein Denaturation in Virus-Host Interactions

Ubiquitination is a vital posttranslational modification that impacts a variety of cellular processes, including the regulation of host-virus interactions and antiviral innate immunity. The ubiquitination of viral proteins plays a role in modulating virus virulence and invasion, while the ubiquitination of host proteins is involved in virus recognition and host-virus interactions, thus influencing antiviral immune responses. Given these diverse functions, there is a growing need to explore the mechanisms underlying protein ubiquitination in the context of host-virus interactions and antiviral innate immunity. Immunoprecipitation after protein denaturation and subsequent immunoblotting with a specific ubiquitination antibody provides a simple and effective method for identifying ubiquitinated proteins during viral infections while minimizing potential interference from interacting proteins. Here, we outline an approach for evaluating protein ubiquitination in HEK 293 T cells during viral infection.