Fig 8.

YscFD28AD46A secretes Yops into the cell supernatant in the presence of HEp-2 cells. A WT, ΔyopN, ΔyopB, or ΔyscF strain expressing pTRC99A, pTRC99A-yscF, or pTRC99A-yscFD28AD46A was grown in 2× YT medium plus 5 mM CaCl₂ for 2 h at 26°C and then grown at 37°C for 2 h. Strains carrying pTRC99A or plasmid derivatives were exposed to IPTG to induce expression of YscF. (A) HEp-2 cells were infected at an MOI of 50:1 (+). Equivalent numbers of bacteria were added to wells lacking HEp-2 cells (−). After a 1-h incubation at 37°C, supernatants were collected, and proteins were analyzed by TCA precipitation and separated by SDS-PAGE. Proteins were visualized by Western blot analysis with anti-YopE antibody. (B) After growth at 37°C, bacteria were gently spun and grown for an additional hour in RPMI medium. Supernatants were collected, and proteins were precipitated with TCA, separated by SDS-PAGE, and visualized by Coomassie blue dye. Each experiment was repeated at least twice, and a representative blot is shown.