VieB is a dose-dependent inhibitor of phosphotransfer. (A) Purified GST-VieS-C was incubated with 32P-ATP-γ either alone (lane 1) or in the presence of equimolar VieA-His6 (lane 2) and 5 μM VieB (lane 3) for 30 minutes at 30°C. (B) GST-VieS-C was incubated with 32P-ATP-γ in the presence of equimolar VieA-His6 and either 0, 0.25, 0.5, 1, 2, 4, or 8 μM VieB for 30 minutes at 30°C. Samples were stopped with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. Proteins labeled with 32P were observed by radioautography. Figure panels are from the same experiment and exposed for the same amount of time. The radioautographs shown are a representative of three replicates. The band just below GST-VieS-C is a VieS degradation band, which is present in all figures and should be ignored.