FIG. 4.

Entry of LcrV into HeLa cells does not require the translocator Yops, YopB and YopD. HeLa cell monolayers were infected, in duplicate, for 4 h at 37°C-5% CO₂ with Y. pestis KIM8-3002.1 (yopB) (lanes 1 to 4) or KIM8-3002.2 (yopD) (lanes 5 to 8) at an MOI of 10. Infection with yopD Y. pestis was done in the presence of cytochalasin D at 5 μg/ml. One replicate of each treatment was treated with trypsin at 100 μg/ml prior to harvest. Fractionation of cultures was done as previously described, and proteins in samples corresponding to 0.3% of original culture volume were resolved in 12% polyacrylamide gels. LcrV and YopE in cell-free medium (M) and soluble (S) HeLa cell water-lysate fractions were detected by immunoblotting with α-HTV and α-YopE. Proteins were visualized by probing with horseradish peroxidase-conjugated anti-rabbit IgG followed by development with ECL reagent.