Figure 2.
Identification of Legionella effector(s) responsible for Rab5 ubiquitination. (a) HeLa-FcγRII cells expressing HA-UB and 3x-FLAG-Rab5A were infected without or with the indicated L. pneumophila Lp02 strains for 1 h at an MOI of 50. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA and FLAG. (b) HeLa-FcγRII cells expressing HA-UB and 3x-FLAG-Rab5A were infected with or without the indicated L. pneumophila Lp01 strains for 1 h at an MOI of 50. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA and FLAG. Data are representative of three independent experiments. Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (c) HeLa-FcγRII cells expressing mRFP-Rab5A were infected with the indicated L. pneumophila Lp01 strains for 1 h at an MOI of 10. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 µm. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P value is determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (d) HeLa-FcγRII cells or the cells expressing mRFP-Rab5A or mRFP-Rab7 were infected with indicated L. pneumophila Lp01 strains for 1 and 2 h at an MOI of 20. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria and then permeabilized and further stained with DAPI for detection of intracellular bacteria or with DAPI and LAMP2 antibody for detection of intracellular bacteria and endogenous LAMP2. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (e) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting Rab7. At 72 h after transfection, the cells were infected with indicated L. pneumophila Lp01 strains for 8 h at an MOI of 5. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. The graph shows the average number of L. pneumophila in a single vacuole. Data are representative of three independent experiments (30 vacuoles were scored in each experiment). Results are shown as means ± SEM. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. *P < 0.05. IB, immunoblotting. Source data are available for this figure: SourceData F2. Refer to the image caption for details.

Identification of Legionella effector(s) responsible for Rab5 ubiquitination. (a) HeLa-FcγRII cells expressing HA-UB and 3x-FLAG-Rab5A were infected without or with the indicated L. pneumophila Lp02 strains for 1 h at an MOI of 50. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA and FLAG. (b) HeLa-FcγRII cells expressing HA-UB and 3x-FLAG-Rab5A were infected with or without the indicated L. pneumophila Lp01 strains for 1 h at an MOI of 50. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA and FLAG. Data are representative of three independent experiments. Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (c) HeLa-FcγRII cells expressing mRFP-Rab5A were infected with the indicated L. pneumophila Lp01 strains for 1 h at an MOI of 10. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 µm. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P value is determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (d) HeLa-FcγRII cells or the cells expressing mRFP-Rab5A or mRFP-Rab7 were infected with indicated L. pneumophila Lp01 strains for 1 and 2 h at an MOI of 20. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria and then permeabilized and further stained with DAPI for detection of intracellular bacteria or with DAPI and LAMP2 antibody for detection of intracellular bacteria and endogenous LAMP2. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. (e) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting Rab7. At 72 h after transfection, the cells were infected with indicated L. pneumophila Lp01 strains for 8 h at an MOI of 5. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. The graph shows the average number of L. pneumophila in a single vacuole. Data are representative of three independent experiments (30 vacuoles were scored in each experiment). Results are shown as means ± SEM. P values are determined using one-way ANOVA with Tukey’s multiple comparisons. *P < 0.05. IB, immunoblotting. Source data are available for this figure: SourceData F2.

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