Extended Data Figure 2. RNF213 is required for LPS ubiquitylation.

a, In vitro ubiquitylation (IVU) of S. Typhimurium Arfc extracted from infected HeLa cells. The reaction comprised FLAG-ubiquitin, E1 enzyme (UBE1), E2 enzyme (UBCH5C), and fractionated HeLa cell lysate as indicated. In the chromatograms depicted below each blot, light grey indicates fractions with little or no LPS-ubiquitylating activity, whereas blue indicates fractions with LPS-ubiquitylating activity used for further fractionation or mass spectrometry.

b,Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and WT HeLa and RNF213^KO HeLa cells c, d, Immunoblot analysis of S. Typhimurium Arfc (c,d left panel) or WT (d right panel) extracted from HeLa cells transfected with the indicated siRNAs (c) or extracted from WT or RNF213^KO MEFs (d). Representative of 3 biological repeats. Blots were probed with the indicated antibodies. Actin and GroEL or DnaK, loading controls for mammalian and bacterial lysates, respectively. *, non-specific band. For gel source data, see Supplementary Fig. 1.