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Fig. 4. (A-C) Immunoblots. (A) Blots from microtubule-binding studies probed with the anti-CrKCBP antibody. Extracts (Ex) were prepared by extracting axonemes with 1 mM ATP and treating with apyrase. The resulting extracts were then added to microtubules (T) assembled in vitro or to an equivalent volume of buffer (B). The microtubules were sedimented, the supernatant collected (S) and the pellet (P) resuspended in an equivalent volume of buffer. In some experiments ATP was added to the resulting pellet (P+ATP) to assess ATP-sensitive binding of CrKCBP to microtubules. (Top panel) Microtubule binding was conducted in the absence of calmodulin under low Ca²⁺ conditions; (second panel) binding was performed in the presence of high Ca²⁺-calmodulin (+Ca²⁺+CaM) using an extract prepared in low Ca²⁺ conditions (-Ca²⁺); (third panel) binding was performed in the presence of Ca²⁺+CaM using an extract prepared in high Ca²⁺ conditions (+Ca²⁺). Microtubule binding of CrKCBP is only mildly sensitive to the presence of Ca²⁺-CaM. (B) Blots from immunoprecipitation experiments using anti-calmodulin antibodies for precipitation and probed with either anti-calmodulin (CaM) or CrKCBP antibodies. Axonemes were extracted with 1 mM ATP in high Ca²⁺ conditions; half of the resulting extract (Ex) was treated with apyrase (+apyrase) and half was not (-apyrase). Precipitations were carried out as described in Materials and Methods and the resulting unbound (un) and precipitated (IP) proteins were processed for gel electrophoresis. The antibodies precipitate virtually all of the calmodulin in the extract but do not precipitate CrKCBP. (C) Blots of sucrose gradient fractions probed with anti-CrKCBP antibodies (fractions 1-23=20-5% sucrose, respectively). CrKCBP sediments at approximately 11S, peaking in fraction 11.

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