IRF3(5D) is deubiquitinated by SARS-CoV PLpro. Plasmids expressing either Flag-tagged IRF3(5D), GFP-tagged PLpro, HA-tagged Ubiquitin, HA-tagged K48 Ubiquitin, or HA-tagged K63 Ubiquitin were transfected into HEK293T cells alone or in combination to assay for ubiquitination statues of IRF3(5D). At 24 hours post transfection, cells were lysed and analyzed by western blot with anti-HA, anti-Flag and anti-GFP. A. A sample (5ul) of each indicated transfection was analyzed by western blot to detect input expression levels. B. Lysate was used for immunoprecipitation with antibody directed against the Flag tag to identify the ubiquitination state of IRF3(5D). Note that IRF3(5D) is ubiquitinated when transfected with HA-tagged K48 or K63 Ubiquitin, however when PLpro is cotransfected with as well, ubiquitination is reduced.