Kinetics of Rab5 on the LCV and characterization of Rab5 ubiquitination upon L. pneumophila infection. (a and b) Images showing typical vacuoles positive or negative for mRFP-Rab5A (a) or mRFP-Rab7 and endogenous LAMP2 (b), as related to Fig. 1. Bar, 5 µm. (c) HEK293-FcγRII cells expressing 3x-FLAG-Rab5A were infected without or with the WT or ∆dotA 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 visualized by silver staining. Asterisks indicate precipitated 3x-FLAG-Rab5A. (d and e) HEK293-FcγRII cells expressing 3x-FLAG-Rab5A (S34N) or (Q79L) (d) or 3x-FLAG-Rab5A (e) were infected without or with the WT or ∆dotA 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 UB and FLAG (d), or K48-specific UB, K63-specific UB, or FLAG (e). (f) HEK293-FcγRII cells were infected with the WT or ∆dotA L. pneumophila Lp01 strains with 10 µg/ml cycloheximide (CHX; 239763-M; Sigma-Aldrich) for the indicated times (hours) to inhibit de novo protein synthesis. After treatment, cell lysates were prepared, and equal amounts of proteins were analyzed by IB with antibodies against α-tubulin and Rab5A. IB, immunoblotting. Source data are available for this figure: SourceData FS1.