Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Subcellular fractionation of AvrPto kinase activity in leaf protein extracts. Six 1-cm diameter N. benthamiana leaf disks were pulverized in 1 mL of extraction buffer (50 mM Tris pH 8.0, 50 mM NaCl, 10 mM EDTA, 5mM DTT, 5uL plant protease inhibitor cocktail (Sigma)). The extract was centrifuged twice at 8000xg for 5 minutes to remove debris, then for 30 minutes at 250,000xg. The supernatant was saved for further analysis, and the pellet washed once with extraction buffer. The pellet fraction was centrifuged again at 250,000xg for 30 min, then resuspended in extraction buffer containing 0.1% Triton-X 100. Each fraction was then tested for kinase activity using AvrPto-FLAG as a substrate. Top panel is SDS-PAGE and Coomassie blue staining of total (T), soluble (S), and pellet (P) fractions. Middle panel is³²P-labeling of AvrPto and lower panel is Coomassie blue staining of ³²P-labeled AvrPto-FLAG showing equal loading.