Fig 2. Measurement of production, translocation and antigen presentation of YopE in vitro.

The indicated strains of Y. pseudotuberculosis were grown at 37°C in LB containing 2.5 mM of CaCl₂ (High Ca²⁺) to inhibit type III secretion (A), or in LB containing 20 mM MgCl and 20 mM NaOX (Low Ca²⁺) to activate type III secretion (B). Lysates of the bacteria were subjected to immunoblotting with anti-YopE antibodies. Immunoblotting with anti-DnaK antibody was used to indicate equal loading. (C) The indicated strains grown at 37°C in low Ca²⁺ LB were used to infect BMDMs at a MOI of 50 for 1.5 h. The infected BMDMs were incubated with a non-iononic detergent buffer and then separated into an insoluble fraction containing bacteria or a soluble fraction containing cytosolic components. Samples of the insoluble (left panel) or soluble (right panel) fractions were subjected to immunoblotting with anti-YopE antibody or anti-β-actin antibody to control for loading. Results shown are representative of three independent experiments. (D) The indicated strains were added to wells without BMDMs or to wells containing BMDMs at MOI of 10 for 4 h, and gentamicin was included during the last 2 h. Then ET-enriched CD8⁺ T cell lines in medium containing penicillin and streptomycin were added to wells containing bacteria alone or to wells containing BMDMs infected with bacteria at 1:4 (BMDM to T cell) ratio and incubated for 48 h before measuring the concentration of IFNγ in the supernatants by ELISA. Data shown are results from one representative of 4 experiments performed.