Depletion of PI(4,5)P2 partially complements Rab5 and PI(3)P localization to sopB mutant SCVs. (A) Cells were cotransfected with Rab5A-GFP and PH(PLCδ)-RFP or synaptojanin-2–CAAX, PH(PLCδ)-RFP, and Rab5A-GFP as indicated. Cells were then infected with either WT or sopB mutant expressing RFP, fixed at 10-min after infection, and stained for extracellular bacteria. Intracellular bacteria colocalizing with Rab5A-GFP was then determined by microscopic analysis. Data are means ± SEM of three separate experiments (>100 bacteria analyzed per experiment). (B) Cells were transfected with the indicated construct and then infected with either WT or sopB mutant labeled covalently with Alexa Fluor 647. Infected cells were analyzed using a spinning disk confocal microscope. Images were acquired at 1-min intervals for ∼1 h. Localization of PI(3)P to SCVs (colocalization with 2FYVE-GFP or PX-mCherry) during the course of infection is shown. Data are means ± SEM of three separate experiments for WT- (94 SCVs analyzed) or sopB mutant– (68 SCVs analyzed) infected cells expressing 2FYVE-GFP. Five separate experiments were analyzed for sopb mutant–infected cells expressing synaptojanin 2-CAAX, PH(PLCδ)-RFP, and 2FYVE-GFP (121 SCVs analyzed), and three separate experiments were analyzed for sopb mutant–infected cells expressing 2PH(PLCδ)-GFP and PX-mCherry-GFP (120 SCVs analyzed). The p-values are shown.
