Figure 4.
Lpg2525 promotes the association of RabGAP-5 with Rab5, and its F-box domain is crucial in recruiting RabGAP-5 to the LCV. (a) HeLa-FcγRII cells were transfected with myc-RabGAP-5, 3x-FLAG Rab5A (Q79L), and GFP, GFP-Lpg2525 (FL) or GFP- Lpg2525 (∆FB). At 24 h after transfection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against myc, GFP, and FLAG. Data are representative of three independent experiments. Results are shown as means ± SD. P-value is determined using one-way ANOVA with Tukey’s multiple comparisons. *P < 0.05. (b) HeLa-FcγRII cells expressing GFP-RabGAP-5 were infected with the indicated L. pneumophila Lp01 strains for the indicated time 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. Bar, 5 µm. Arrows indicate the position of intracellular L. pneumophila. 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. (c) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting RabGAP-5. At 48 h after transfection, the cells were transfected with an additional plasmid-encoding mRFP-Rab5A for 24 h, infected with the WT L. pneumophila Lp01 strain 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. 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. IB, immunoblotting. Source data are available for this figure: SourceData F4. Refer to the image caption for details.

Lpg2525 promotes the association of RabGAP-5 with Rab5, and its F-box domain is crucial in recruiting RabGAP-5 to the LCV. (a) HeLa-FcγRII cells were transfected with myc-RabGAP-5, 3x-FLAG Rab5A (Q79L), and GFP, GFP-Lpg2525 (FL) or GFP- Lpg2525 (∆FB). At 24 h after transfection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against myc, GFP, and FLAG. Data are representative of three independent experiments. Results are shown as means ± SD. P-value is determined using one-way ANOVA with Tukey’s multiple comparisons. *P < 0.05. (b) HeLa-FcγRII cells expressing GFP-RabGAP-5 were infected with the indicated L. pneumophila Lp01 strains for the indicated time 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. Bar, 5 µm. Arrows indicate the position of intracellular L. pneumophila. 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. (c) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting RabGAP-5. At 48 h after transfection, the cells were transfected with an additional plasmid-encoding mRFP-Rab5A for 24 h, infected with the WT L. pneumophila Lp01 strain 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. 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. IB, immunoblotting. Source data are available for this figure: SourceData F4.

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