Extended Data Figure 4: IpaH9.8 directly targets hGBP1 for K48-linked polyubiquitination on multiple lysine residues.

a, Lysates of 293T cells co-transfected with Flag–hGBP1 and Myc–IpaH9.8, Myc–IpaH3 or Myc–IpaH7.8 were immunoprecipitated with Flag antibody and immunoblotted as shown. Synthetic Lys48 (K48)- and Lys63 (K63)-linked ubiquitin chains or linear tetra-ubiquitin were included as controls for the linkage-specific antibodies. b, Gel filtration chromatography of purified hGBP1 and IpaH9.8. Coomassie blue-stained SDS–PAGE gel of fractions eluted from the Superdex 200 column. c, Effects of overexpression of mCherry–IpaH9.8-C337A on GFP–hGBP1 recruitment to intracellular S. flexneri ΔipaH9.8 in HeLa cells. Fluorescence images taken 2 h after infection (nearly all cells were successfully infected by the bacteria). Scale bar, 10 μm. d, In vitro ubiquitination of hGBP1 by IpaH9.8 and other IpaH-family proteins. Coomassie blue-stained SDS–PAGE gel shows the Flag–hGBP1 and IpaH proteins added to the reaction. e, f, Ubiquitination sites in hGBP1. The lysine residues indicated in e, except for Lys207 and Lys209, were identified by mass spectrometry analyses of in vitro ubiquitinated hGBP1 (the seven residues labelled in red were identified after the other labelled residues were mutated to arginine (8KR)). 15KR is a mutant hGBP1 in which all fifteen lysine residues have been replaced with arginine. f, 293T cells were co-transfected with Flag–hGBP1 (WT, 8KR or 15KR) and 6×Myc–IpaH9.8. Cell lysates were immunoprecipitated with Flag antibody and the immunoprecipitates were immunoblotted as shown. C/A, hGBP1-C337A. All data shown are representative of three independent experiments.