Figure 3

Y. pestis strain KIM8-3002 (wt) (lane 1 and lanes 3–6) and KIM8-3002.2 (ΔyopD) (lane 2 and lanes 7–10) both containing plasmid pYopE₁₂₉-Elk were used to infect HeLa cells at an MOI of 10. 4 h following infection the culture supernatant containing non-adherent bacteria were removed the remaining adherent HeLa cells were solubilized in 2X SDS-PAGE buffer. Following solubilization proteins were separated and immunoblotted to triplicate PVDF membranes. The triplicate blots were probed with α-YopE, α-Elk, or α-PO₄-Elk, followed by incubation with an alkaline phosphatase conjugated secondary antibody and developed using NBT/BCIP. To some samples anti-sera specific for HT-YscF (lanes 3–4 and lanes 7–8) or the Pseudomonas protein, PcrG (lanes 5–6 and lanes 9–10), were added at dilutions of 1:10 (lanes 3, 5, 7, and 9) or 1:25 (lanes 4, 6, 8, and 10).