Figure 1.
Rab5 ubiquitination upon L. pneumophila infection. (a) HeLa-FcγRII cells expressing mRFP-Rab5A were infected with WT or ∆dotA L. pneumophila Lp01 strains for the indicated times 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. (b) HeLa-FcγRII cells or the cells expressing mRFP-Rab5 or mRFP-Rab7 were infected with WT or ∆dotAL. pneumophila Lp01 strains for the indicated times 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 the 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. **P < 0.01. *P < 0.05. (c) HEK293-FcγRII cells expressing 3x-FLAG-Rab5A were infected without or with WT or ∆dotA L. pneumophila Lp01 strains for 1 h at the indicated MOI. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against UB or FLAG. (d–f) HEK293-FcγRII cells expressing HA-UB (WT) and 3x-FLAG-Rab5A (d), HA-UB (WT) and 3x-FLAG-Rab5A (S34N), or 3x-FLAG-Rab5A (Q79L) (e), or HA-UB (K48R) or HA-UB (K63R) and 3x-FLAG-Rab5A (f) were infected without or with WT or ∆dotA (d only) L. pneumophila 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. IB, immunoblotting. Source data are available for this figure: SourceData F1. Refer to the image caption for details.

Rab5 ubiquitination upon L. pneumophila infection. (a) HeLa-FcγRII cells expressing mRFP-Rab5A were infected with WT or ∆dotA L. pneumophila Lp01 strains for the indicated times 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. (b) HeLa-FcγRII cells or the cells expressing mRFP-Rab5 or mRFP-Rab7 were infected with WT or ∆dotAL. pneumophila Lp01 strains for the indicated times 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 the 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. **P < 0.01. *P < 0.05. (c) HEK293-FcγRII cells expressing 3x-FLAG-Rab5A were infected without or with WT or ∆dotA L. pneumophila Lp01 strains for 1 h at the indicated MOI. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against UB or FLAG. (d–f) HEK293-FcγRII cells expressing HA-UB (WT) and 3x-FLAG-Rab5A (d), HA-UB (WT) and 3x-FLAG-Rab5A (S34N), or 3x-FLAG-Rab5A (Q79L) (e), or HA-UB (K48R) or HA-UB (K63R) and 3x-FLAG-Rab5A (f) were infected without or with WT or ∆dotA (d only) L. pneumophila 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. IB, immunoblotting. Source data are available for this figure: SourceData F1.

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