FIG. 5.

HT-VN68 is not secreted by Y. pestis and cannot complement Yop targeting. Y. pestis KIM5-3241 (lcrV yopJ) (lane 1) or Y. pestis KIM5-3241 carrying pHT-V (lanes 2 to 5) or pHT-VN68 (lanes 6 to 9) was used to infect HeLa cells, in duplicate, at an MOI of 10 in the presence of 0.1 mM IPTG. After 4 h, one replicate for each was treated with trypsin (lanes 4, 5, 8, and 9) at 100 μg/ml to assess protease accessibility of HT-V, HT-VN68, and YopE. The presence of proteins in cell-free medium (M) or H₂O-released HeLa cell soluble (S) fractions was analyzed by resolving samples representing 20% of the original cultures in a 12% polyacrylamide gel followed by immunoblotting. Blots were probed with α-HTV and α-YopE, and proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies followed by development with NBT-BCIP.