FIG. 3.

Stability and expression analysis of chaperone hybrids and their effect on substrate stability. (A) The intrabacterial stability of chaperone hybrids (left panel) and YopD substrate (right panel) produced by Y. pseudotuberculosis grown at 37°C in BHI supplemented with 2.5 mM CaCl₂ (type III secretion repressed) and 0.4 mM IPTG was examined. At time zero, chloramphenicol was added in order to stop de novo protein synthesis. Samples from pelleted bacteria were taken at different time intervals, and the amount of protein was detected by Western blot. (B) Immunoblot of synthesized chaperone hybrids prepared from pelleted bacteria grown in Yop inducing medium (BHI minus Ca²⁺) supplemented with 0.4 mM IPTG. Hybrids were identified using monoclonal anti-FLAG antiserum and YopD by a polyclonal rabbit anti-YopD antiserum.