Figure 5

CesT membrane association is reduced in the absence or with limited EscU auto-cleavage. (A) Total cell lysates and membrane fractions were probed with anti-CesT antibodies to assess CesT protein levels. The membrane fraction immunoblot was subjected to quantification of band intensity (chemiluminescent signals) to measure CesT protein levels relative to EscJ. EscJ forms a multimeric ring like structure (independent of EscU) and localizes to the inner membrane. For quantification via densitometry, the immunoblots were probed with antibodies and then simultaneously imaged using an exposure time within an empirically determined linear range of signal detection. The densitometry values are averages from three independent experiments and are expressed as a ratio of CesT/EscJ signals as assayed by Quantity One software. A dependent, match paired student's t test was used to assess statistical significance between values (denoted by an asterisk). A representative immunoblot from the experiments is shown. (B) Sucrose density gradient fractionation of membrane preparations from the indicated strains. EscJ and intimin are known inner and outer membrane proteins and their immune-detection served to indicate fractions enriched for inner and outer membranes separated upon ultracentrifugation. Note the altered distribution of CesT in the presence of EscU(N262A) and EscU(P263A).