VieB is not a phosphatase or stimulates GST-VieS-C phosphates activity. (A) Previously described phosphotransfer assays were conducted at 30°C in the presence of 32P-ATP-γ with the following modifications: Lane 1- GST-VieS-C alone was incubated for 60 minutes, Lane 2- GST-VieS-C was incubated with 1 μM VieA-His6 for 60 minutes, Lane 3- GST-VieS-C was incubated with 1 μM VieA-His6 for 30 minutes then 5 μM wild-type VieB was added for an additional 30 minutes, Lane 4- GST-VieS-C was incubated with 5 μM wild-type VieB for 30 minutes then 1 μM VieA-His6 was added for an additional 30 minutes. Proteins labeled with 32P were observed by radioautography. The radioautographs shown are a representative of three replicates. (B)
32P-VieA-His6 was incubated with buffer, GST-VieS-C alone or GST-VieS-C and VieB for 60 minutes at 30°C. Samples were taken over time and quantified. 0 minute time point represents 100% 32P-VieA-His6. Error bars represent the SEM of three replicates.