Figure 3.

tmRNA-tagged proteins in ΔhemK and other cells were detected by immunoblot analysis. Chromosomal ssrA in each strain (lane 1, WK103; lane 2, W3110; lane 3, CA274; lane 4, LE392; lane 5, C600) was inactivated by transducing ΔssrA∷Km, and a plasmid encoding the FLAG-tagging ssrA variant was introduced. Then, cells grown in LB broth were harvested at OD₆₀₀ of about 0.7, suspended in SDS sample buffer (200 μl/OD₆₀₀-unit, and boiled for 5 min, and 10 μl of each cell extract was then analyzed by SDS/PAGE (12% acrylamide) and immunoblotting with anti-FLAG antibody.