Fig. 6. Pelle is transphosphorylated and capable of self-association. (A) GST-PelleK240R is phosphorylated by CIP-Pelle. GST-PelleK240R and/or CIP-Pelle was incubated in kinase buffer containing [-³²P]ATP as in Fig. 4. Proteins were analyzed by SDS-PAGE as in Fig. 5A. Lane 1, 250 ng CIP-Pelle; lane 2, 250 ng CIP-Pelle and 500 ng GST-PelleK240R; and lane 3, 500 ng GST-PelleK240R. (B) Pelle self-association. Purified GST or GST-Pelle (2 µg) was immobilized on glutathione beads, and incubated with 1 µg of purified CIP-Pelle. Protein complexes were eluted with 20 mM glutathione (reduced form) and analyzed by SDS-PAGE followed by anti-Pelle immunoblot. Lane 1, 20% of CIP-Pelle used in binding assays (input); lanes 2 and 3, GST or GST-PelleK240R, respectively, incubated with CIP-Pelle.