VieB does not affect the stability of phosphorylated GST-VieS-C. Purified GST-VieS-C or MBP-VieS-C D1018A was incubated with 32P-ATP-γ in the absence of 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 buffer or with 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 (A, B) and quantified in (C).The radioautograph shown is a representative of three replicates. Error bars represent the SEM of three replicates.