Fig. 5. K236 is the site of UBE2O-mediated ubiquitination in IFIT3.

A Plasmids containing different tags were cotransfected into 293 T cells. Immunoprecipitation using an anti-Myc antibody, followed by immunoblotting (IB) with the corresponding antibodies, was conducted to determine the type of ubiquitination (n = 3). B Prediction of ubiquitination sites in IFIT3 using the CKSAAP database. C Prediction of ubiquitination sites in IFIT3 using the GPS-Uber database. D IFIT3 mutants were generated based on prediction results from the CKSAAP database and transfected into 293 T cells for the in vivo ubiquitination assay. Mutation of K236 abolished the ubiquitination-mediated degradation of IFIT3 via UBE2O. E Based on the prediction results from the GPS-Uber database, mutations were introduced into IFIT3, and plasmids with the appropriate tags were transfected. IP was performed using an anti-Myc antibody, while IB was carried out using the corresponding antibody. Mutation of K236 abolished the ubiquitination-mediated degradation of IFIT3.