Fig. 5. CIP-Pelle can be re-autophosphorylated and activated in a concentration-dependent manner in vitro. (A) CIP-Pelle was first incubated in kinase buffer at the indicated concentrations, at 30°C for 1 hour. Identical amounts of pre-incubated Pelle were then removed and incubated in kinase buffer at an identical concentration (5 µg/ml) with 5 µg/ml of HisTubeN at 30°C for 30 minutes. Reaction mixtures were adjusted to contain identical concentrations of buffer components and ATP. Identical aliquots were loaded onto an SDS gel and analyzed as in Fig. 4B. The different CIP-Pelle concentrations during preincubation are indicated: lanes 1, 2 and 3 contained CIP-Pelle at 25, 50 and 100 µg/ml, respectively. (B) CIP-Pelle and HisTubeN in the final reaction mixtures analyzed in Fig. 5A were detected by western blotting with anti-Pelle and anti-His antibodies, respectively. Two exposures of the anti-Pelle blot are shown and the positions of distinct Pelle isoforms are indicated.