For each reaction, 1.8 × 108 cells producing the wild type AcrA from the chromosome (WT) or the plasmid (WT(plasmid)) and the plasmid-borne AcrA with indicated double cysteine substitutions were treated with trypsin at the final concentrations of 0.0, 0.1, 1.0 and 10.0 μg/ml. Total proteins were separated by 12% SDS-PAGE and probed with polyclonal anti-AcrA antibodies. The major proteolytic fragments of AcrA assembled into the complex, Q29-K374 and T47-R315, are indicated by arrows.