Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery
Fig 5
Identification of host SHOC2 and PSMC1 as conserved targets of IpaH4.
A. Representative immunoblot of in vitro ubiquitination assay performed with indicated GST-IpaH proteins in the absence of substrates. B. Representative immunoblot of in vitro ubiquitination assay performed with indicated concentrations of wild-type GST-IpaH4 or GST-IpaH4C339S mutant proteins. C. Schematic outlining UBAIT protocol [53,56]. D. Venn diagram showing conserved putative substrates (overlapping region) of IpaH4 across UBAIT experiments (n = 3) in LD652 cell lysates and Y2H screens (n = 2) against a human prey library. E. Representative immunoblot of in vitro ubiquitination assay showing IpaH4-mediated ubiquitination of human Flag-SHOC2 proteins. F. Representative immunoblot of in vitro ubiquitination assay showing IpaH4-mediated ubiquitination of human PSMC1-His proteins. G. Representative immunoblot of degradation assays using indicated Flag-tagged human proteins in transfected HEK293T cells co-expressing GFP, IpaH4 (WT) or catalytic mutant GFP-IpaH4C339S (C339S). H. Representative immunoblot of degradation assays using indicated Flag-tagged human proteins in transfected LD652 cells co-expressing GFP, IpaH4 (WT) or catalytic mutant GFP-IpaH4C339S (C339S). I. Representative immunoblot of degradation assays of Flag-tagged moth (L. dispar) protein in LD652 cells expressing GFP, IpaH4 (WT) or catalytic mutant GFP-IpaH4C339S (C339S). Images in G-I were created with BioRender.com.