Fig. 1.

Purification of active ROP18 kinase proteins. A, schematic illustration of the GST-tagged ROP18 constructs GST-ROP18^Δ83 and GST-ROP18^Δ83-KD. ROP18^Δ83 lacks the signal peptide and the prodomain, and the kinase-dead mutant ROP18^Δ83-KD is a D394A-mutated version of ROP18^Δ83. B, GST-ROP18^Δ83 and GST-ROP18^Δ83-KD proteins were expressed in yeast and purified using glutathione beads. The purified proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. C, purified GST-ROP18^Δ83, GST-ROP18^Δ83-KD, and EGFR (positive control) were incubated with kinase assay buffer containing [γ-³²P]ATP. Proteins were then dotted on nitrocellulose membrane, followed by autoradiography.