FIG. 6.

Both the N and C termini of LcrV are required to mediate Yop targeting and for release of LcrV from bacteria. Y. pestis KIM8-3002 (parent), KIM5-3241 (lcrV yopJ), and KIM8-3002.8 (lcrG lcrV) alone (lanes 1, 2, 7, 8, 13, and 14), carrying pVC216 (lanes 5, 6, 11, 12, 17, and 18) or carrying pE15VN68 (lanes 3, 4, 9, 10, 15, and 16) were used to infect HeLa cells, in duplicate, at an MOI of 10 in the presence of 0.2 mM IPTG. After 4 h, trypsin was added to replicate wells at 100 μg/ml to assess protease accessibility of LcrV and YopE. Samples were fractionated, and portions representing 12% of the original cultures were resolved in 12% polyacrylamide gels. Native LcrV, truncated LcrVs, and YopE in the HeLa cell soluble fraction (A) or released into culture medium (B) were detected by probing blots with α-HTV and α-YopE. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies followed by development with NBT-BCIP.