Fig. 2: IpaH1.4/2.5 bind to, ubiquitylate and antagonize RNF213.
From: Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213
a, HeLa cell lysate incubated with beads displaying the indicated GST-tagged IpaH proteins. Bound RNF213 was detected by western blot. The results are representative of n = 3 experiments. b, Immunoblot analysis of Salmonella Typhimurium ΔrfaL, extracted from HeLa cells expressing the indicated GFP-tagged IpaH proteins. Gray triangle, ΔrfaL LPS; black circle, ubiquitin; GroEL, loading control for bacterial lysates; cd, catalytically dead (E3 inactive) variants; actin, loading control for HeLa lysates. The LPS fractions were isolated by heat clearance of bacterial lysates and probed for conjugated ubiquitin with FK2 antibody. The loading control (GroEL) was probed for in non-heat-cleared bacterial lysates. All other proteins were probed for in host cell lysates. The results are representative of n = 4 experiments. c, Coomassie-stained gel of an in vitro ubiquitylation reaction containing Flag-tagged ubiquitin, UBE1, UBCH5C, the indicated IpaH proteins and enzymatically inactive RNF213-H4509A (Extended Data Fig. 2a) as substrate. The majority of RNF213 is ubiquitylated by IpaH1.4/2.5, yielding a product of substantially increased molecular mass that migrates more slowly in the gel, as indicated. Bottom, immunoblot for Flag-tagged ubiquitin. The results are representative of n = 3 experiments.
