Figure 3. In vivo steady-state protein levels of wild-type and CphA mutants.

Steady-state protein levels of StrepII-tagged wild-type and CphA mutants in recombinant E. coli were determined by SDS-PAGE of whole cell lysates followed by immunoblotting with an anti-StrepII tag monoclonal antibody conjugated to horseradish peroxidase (HRP). Constitutively expressed DnaK (~70 kDa) was used as a loading control and probed with anti-DnaK monoclonal antibody and HRP-conjugated secondary antibody. The hybridization signal for wild-type and mutant CphA-StrepII and DnaK was quantified by densitometry. The signal for CphA-StrepII was normalized to that for DnaK in the same sample. Protein levels of mutant CphA-StrepII are expressed in the bar graph relative to that of the wild-type protein, which was set as 1. Quantification data are based on three independent experiments and a representative blot is shown.