Fig. 4. Autophosphorylation is necessary for Pelle kinase activity. (A) Both autophosphorylated HisPelle (lane 2) and dephosphorylated CIP-Pelle (lane 1) were expressed in and purified from E. coli (see Materials and Methods), and subjected to SDS-PAGE followed by anti-Pelle western blotting. (B) Different forms of Pelle were incubated in kinase buffer containing [-³²P]ATP, with or without HisTubeN, at 30°C for 30 minutes. Reactions were terminated by TCA precipitation, and precipitates were washed and subjected to SDS-PAGE. Phosphate incorporation was analyzed by autoradiography. Top: lane 1, 100 ng of HisTubeN; lane 2, 200 ng of CIP-Pelle; lane 3, 100 ng of HisTubeN plus 200 ng of CIP-Pelle; lane 4, 200 ng of HisPelle; and lane 5, 100 ng of HisTubeN and 200 ng of HisPelle. There were equivalent amounts of Pelle (anti-Pelle blot) and HisTubeN (anti-His blot) in lanes 3 and 5 (bottom).