Fig. 1: The S. flexneri effector IpaH1.4 can directly interact with RNF213.

a A schematic diagram showing the strategy used for the identification of IpaH1.4-binding proteins in HEK293F cells based on a biochemical affinity purification coupled with mass spectrometric analyses. b The volcano plot illustrating the comparison of FLAG pull-downs from HEK293F cell lysates (n = 3) overexpressing the FLAG-TEV-IpaH1.4 C368A mutant with the empty plasmid vector FLAG-TEV serving as the control in the affinity purification coupled to mass spectrometry (AP-MS) analyses. Notably, a two-tailed unpaired Student’s t test analysis was performed for the analysis of this AP-MS data. Proteins significantly enriched in the anti-FLAG bead pull-downs are annotated for clarity (p value < 0.05, fold change >32; except for HOIP). The identified RNF213 is highlighted in red, HOIP and HOIL-1L are highlight in purple, and IpaH1.4 is highlighted in blue. c GST pull-down assays of the GST-tagged IpaH1.4 or the GST tag with the FLAG-tagged full-length human RNF213 expressed in HEK293F cells. This experiment was independently repeated twice. d Co-immunoprecipitation assays showing the mCherry-tagged full-length IpaH1.4 can interact with the AcGFP-tagged human full-length RNF213 and the RNF213(371-5207) fragment in HEK293T cells. IP immunoprecipitation, IB immunoblotting. This experiment was independently repeated twice. e Size exclusion chromatography (SEC)-based analysis of the interaction between RNF213(371-5207) and IpaH1.4(1-575) performed on the Superose 6 increase 10/300 GL column. In this panel, “SUM” stands for the theoretical sum of RNF213(371-5207) and IpaH1.4(1-575) profiles, and A280 stands for the absorbance at 280 nm. For the reconstitution of the binding between RNF213(371-5207) and IpaH1.4(1-575), the RNF213 and IpaH1.4 proteins were expressed and purified separately, and then mixed together prior to the (SEC)-based analysis. f SDS-PAGE combined with Coomassie blue staining analyses showing the protein components of the corresponding fraction 12 to 17 collected from the SEC-based experiments in (e).