tra12158-sup-0001-FigureS1.docWord document256KFigure S1: Replicative vacuole assay in NLRC4−/− cells. NLRC4−/− bone marrow-derived macrophages were infected with L. pneumophila wild-type, ΔdotA and ΔsdcA-sidC strains for 1 h (A) or 8 h (B). Cells were fixed and stained with antibodies against ubiquitin (red) or Legionella (green). Nuclei were stained with Hoechst. C) Quantification of ubiquitin-positive vacuoles after 1 and 8 h. Average ± standard deviations were obtained from three independent experiments in which 300 vacuoles were scored for each strain. Statistical significance was obtained by comparing the number of cells showing ubiquitin-positive bacteria at 1 and 8 h between WT and ΔsdcA-sidC mutant (*p < 0.01).
tra12158-sup-0002-FigureS2.docWord document380KFigure S2: Single deletion mutants ΔsidC and ΔsdcA are not impaired for Arf1 recruitment. HEK293 cells stably expressing GFP-tagged Arf1 were infected with L. pneumophila wild-type, ΔsdcA, ΔsidC and ΔsidC_sdcA. A) Cells were fixed 1 h post-infection. Intracellular bacteria are shown in red. B) Quantification of the proportion of Arf1-positive vacuoles. Average ± standard deviations were obtained from three independent experiments (*p < 0.01).
tra12158-sup-0003-FigureS3.docWord document65KFigure S3: Rab1 is monoubiquitinated. HEK293 cells stably expressing Flag-tagged Rab1 were either uninfected or infected with L. pneumophila wild-type or ΔdotA for 1 h, following which they were lysed. FLAG-tagged Rab1 was immunoprecipitated using FLAG antibody-coated beads. Immunoprecipitated proteins were boiled and subjected to SDS–PAGE analysis and subsequently probed with either a Rab1A antibody or an ubiquitin-specific antibody FK2. The monoubiquitinated Rab1 band has been marked with an arrow in both blots.
tra12158-sup-0004-FigureS4.docWord document115KFigure S4: Dimeric SidCNT assembly found in the crystal. One SidCNT is shown in surface representation and the second SidCNT in ribbon representation.
tra12158-sup-0005-FigureS5.docWord document146KFigure S5: Analytical size exclusion chromatography of SidC shows that SidC is a monomer in solution. Analytical size exclusion chromatography (Superdex S75 10/30) of SidCFL, SidCΔC and SidCNT shows that SidC is a monomer in solution.

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