Figure 3.
Lpg2525 forms a complex with SKP1–Cullin1, and the F-box domain of Lpg2525 is required for Rab5 ubiquitination. (a) HeLa-FcγRII cells were transfected with a plasmid-encoding 3x-FLAG vector, 3x-FLAG-Lpg2525 (FL), or 3x-FLAG-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 Cullin1, SKP1, and FLAG. (b) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting SKP1. At 48 h after transfection, the cells were transfected with an additional plasmid-encoding 3x-FLAG-Lpg2525 (FL) for 24 h, lysed, and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with the indicated antibodies. (c) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (Q79L), HA-UB, 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 GFP, FLAG, and HA. (d) HeLa-FcγRII cells expressing 3x-FLAG-Rab5A and HA-UB were infected with 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 < 0.05. (e) HeLa-FcγRII cells or the cells expressing mRFP-Rab5A or Rab7 were infected with the 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. Phagocytic markers positive LCVs were quantified, and the representative data of three independent experiments were presented (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 < 0.001. IB, immunoblotting. Source data are available for this figure: SourceData F3. Refer to the image caption for details.

Lpg2525 forms a complex with SKP1–Cullin1, and the F-box domain of Lpg2525 is required for Rab5 ubiquitination. (a) HeLa-FcγRII cells were transfected with a plasmid-encoding 3x-FLAG vector, 3x-FLAG-Lpg2525 (FL), or 3x-FLAG-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 Cullin1, SKP1, and FLAG. (b) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting SKP1. At 48 h after transfection, the cells were transfected with an additional plasmid-encoding 3x-FLAG-Lpg2525 (FL) for 24 h, lysed, and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with the indicated antibodies. (c) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (Q79L), HA-UB, 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 GFP, FLAG, and HA. (d) HeLa-FcγRII cells expressing 3x-FLAG-Rab5A and HA-UB were infected with 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 < 0.05. (e) HeLa-FcγRII cells or the cells expressing mRFP-Rab5A or Rab7 were infected with the 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. Phagocytic markers positive LCVs were quantified, and the representative data of three independent experiments were presented (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 < 0.001. IB, immunoblotting. Source data are available for this figure: SourceData F3.

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