Figure 5. YopT Cleavage Does Not Prevent Rac1 Activation.

COS1 cells were cotransfected with plasmids expressing mYFP-PBD and either mCFP-Rac1 or mCFP-Rac1C189S followed by exposure to Y. pseudotuberculosis for 30 min (Materials and Methods) and FRET analysis in cytoplasmic ROIs.

(A–C) Cells expressing mCFP-Rac1(WT) challenged with either YP17 (control) (A) or mCFP-Rac1(WT) challenged with YP17/pYopT (+YopT) (B). Rac1 activation is maintained in the presence of YopT cleavage (C). Data for normalized Rac1 activity from eight cells described in (A) and (B) were quantitated as mean ± SEM.

(D) Cytoplasmic localization of Rac1 does not affect activation. FRET was analyzed in ROIs from cytoplasmic regions of uninfected cells transfected with mCFP-Rac1(WT) or mCFP-Rac1C189S and normalized versus amount of mCFP observed in each cell. NS, no significant difference between control and experimental.

(E) Example of normalized FRET observed in uninfected cell expressing mCFP-Rac1C189S. White bar represents 10 μm.