Figure 5.

Insertion of PopB and PopD into cell membranes in the presence of P. aeruginosa. A, PopB and PopD were detected on HeLa cell membranes after incubations with PAK at physiological pH. The PAKΔexsEΔexoSTYΔpopD::popD strain was incubated with HeLa cells in the indicated medium for 1 h. After the incubation, secreted proteins in 1 ml of medium were collected by centrifugation, precipitated with TCA, and analyzed by immunoblotting using anti-PopB and anti-PopD antibodies as described under “Experimental procedures.” Infected HeLa cells were lysed with 0.1% Triton X-100, and Triton-solubilized proteins were separated from residual bacteria and insoluble debris using centrifugation. 20 μg of total protein in each Triton-solubilized protein sample was analyzed for the presence of PopB and PopD using immunoblotting. B, translocators secreted to the medium cannot bind to HeLa cells. HeLa cells alone (lane 1), incubated with PAK (lane 2), or incubated with the supernatant containing secreted proteins (lane 3) were collected and analyzed as described in A. The supernatant containing secreted PopB and PopD was obtained by centrifugation after culturing PAK with HeLa cells in DMEM plus FBS for 1 h. Representative blots from two independent experiments are shown.