Fig. 4.

The PIF domain is required for PDK1-mediated PID phosphorylation in vitro and in vivo. (A) Binding of PDK1 to PID and PDF protein was visualized by Western blotting as described in the legend for Fig. 3. (B) PID phosphorylation level was compared with that of PDF in the absence or presence of bound PDK1. (C) Equivalent amounts of GST:PID or GST:PDF proteins were incubated with seedling shoot extract in kinase buffer in the presence of [γ-³²P]ATP for 30 min. Protein phosphorylation was visualized by autoradiography. All experiments were performed in triplicate. Autoradiographs were exposed for 1 h in B and 12 h in C.