VieB inhibits VieS intra-molecular phosphotransfer. Purified GST-VieS-C was incubated with 32P-ATP-γ in the absence of VieA-His6 and VieB for 30 minutes at 30°C. Excess 32P-ATP-γ was removed from phosphorylated VieS-C constructs by gel filtration. P-VieS-C constructs were then incubated with additional MnCl2 and either buffer, 1 μM VieA-His6 or pre-mixed 1 μM VieA-His6 and 5 μM VieB for 60 minutes. Samples were stopped at indicated time points with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. VieS-C constructs labeled with 32P were observed by radioautography. The radioautograph shown is a representative of three replicates.