FIG. 3.

Stability of TonB under steady-state conditions. All strains were grown to mid-exponential phase in LB broth supplemented with 100 μg ml⁻¹ ampicillin. Chloramphenicol was then added to a final concentration of 100 μg ml⁻¹ to halt protein synthesis. Samples were taken at 0, 15, 30, 60, and 120 min and precipitated in 10% (wt/vol) trichloroacetic acid. Samples were washed in 100 mM Tris-Cl (pH 8.0) and then suspended in 25 μl of Laemmli sample buffer, incubated at 98°C for 5 min, and resolved on sodium dodecyl sulfate-11% polyacrylamide gels, and a subsequent immunoblot analysis was performed as described in the legend to Fig. 1. Strains are identified by their relevant phenotypes on the left, sample times (in minutes) are indicated at the top, and the positions of molecular mass standards are indicated on the right. B, ExbB; D, ExbD; Q, TolQ; R, TolR.