Fig. 2. The N-terminal domain of pEg2 does not affect the activity of the full-length kinase and does not bind to XlEg5. (A) The pEg2 kinase activity of the recombinant pEg2-(His)6 protein was assayed in vitro using myelin basic protein (MBP) as a substrate in the presence of increasing amounts of either the recombinant inactive pEg2-K/R-(His)6 kinase or the recombinant N-terminal domain Nt-pEg2-(His)6 protein. After incubation in the presence of [-³²P] ATP, the reaction mixture was subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose membrane. The radioactive MBP was counted with a Phosphoimager. The kinase activity is expressed as a percentage of the activity without inhibitor. The kinase activity was estimated in the presence of the inactive kinase (filled circles) or the N-terminal domain of pEg2 (open circles). (B) Affinity chromatography. A Ni-NTA agarose column saturated with either Fl-pEg2-(His)6 (full length), Nt-pEg2-(His)6 (N-terminal) or no protein (control) was loaded with 200 µl of Xenopus CSF extract. Affinity-bound proteins in the different columns were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. The membranes were cut and the upper part (>70 kDa) was incubated with anti-XlEg5 polyclonal antibodies (dilution 1/1000), while the lower part (<70 kDa) was incubated with the anti-pEg2 1C1 mAb (diluted 1/100). Lane 1, Xenopus egg extracts; lanes 2-5, elution fractions; Fl-pEg2-(His)6 column, nickel column preloaded with the full-length pEg2-(His)6 recombinant protein; Nt-pEg2-(His)6 column, nickel column preloaded with pEg2 N-terminal-(His)6 recombinant protein; Control column, control nickel column without any recombinant protein.

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