FIG. 2.
Coimmunoprecipitation of YaeT with histidine-tagged YfgL to establish the specificity of the histidine antibody and the coimmunoprecipitation procedure. (A) Whole-cell lysates were immunoprecipitated with (+) or without (−) monoclonal anti-His antibody. The collected immunoprecipitates were separated by SDS-PAGE and visualized by silver staining. Lane 1, production of plasmid-borne histidine-tagged AcrA (AcrA6His), a component of the TolC-AcrAB tripartite efflux system (9) in E. coli; lanes 2 and 3, YfgL-His6 (YfgL6His) was produced from plasmid pBAD24 and YaeT from the chromosome; lanes 4 and 5, yfgL-His6 was expressed from pBAD24, and yaeT was expressed from both the chromosome and plasmid pBAD33 (12). Hc, immunoglobulin G heavy chain; Lc, immunoglobulin G light chain. (B) Histidine-tagged AcrA or YfgL in the corresponding immunoprecipitate eluates was confirmed by Western analysis using HisProbe-HRP.