1:30,000 in TBST for 30 min. The ECL Plus Western Blotting kit (GE Health- care) was used for detection. Microtubule sliding Flagella were severed from cell bodies by the dibucaine method (Witman, 1986) and isolated by differential centrifugation in buffer A (10 mM Hepes, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, and 50 mM potassium acetate). Axonemes were isolated by adding NP-40 (Calbiochem) to ﬂagella for a ﬁnal concentration of 0.5% (wt/vol) to remove ﬂagellar membranes. Measurement of sliding velocity between doublet microtubules was based on the methods of Okagaki and Kamiya (1986). Microtubule sliding was initiated with buffer A containing 1 mM ATP and 2 μg/ml type VIII protease (Sigma-Aldrich) and was recorded as described previously (Smith, 2002a). All data are presented as mean ± SEM. The t test was used to determine the signiﬁcance of differences between means. For some experiments, axonemes were incubated with afﬁnity-puriﬁed anti–CaM-IP2 antibodies for 15 min at room temperature (22°C) before the induction of sliding. Antibody concentrations are noted on corresponding ﬁgures and are estimates based on the conversion factor of IgG = 150,000 g/mol. Online supplemental material Fig. S1 shows an amino acid sequence comparison of CaM-IP2 and human AAT-1. Fig. S2 shows Northern blots comparing transcript levels of CaM-IP2 and RSP3 before and after deﬂagellation. Fig. S3 shows Western blots of sucrose gradient fractions and immunoprecipitation experiments of corresponding pooled fractions. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200703107/DC1. We gratefully acknowledge Winﬁeld Sale for the gift of antibodies and strains, Dennis Diener for the gift of antibodies, Heather Benson for the preparation of Fig. 4 A, and Laura Fox for technical assistance with Fig. 7. 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