In Ciona, immunoprecipitation was performed as described previously (51) with slight modifications. Anti-DYBLUP antiserum was diluted 1:25 with a binding buffer containing 1.5 M glycine and 3 M NaCl (pH 8.9). The diluted antiserum was added to Protein A Sepharose beads (GE Healthcare) and mixed on a rotator for 4 hours at 4°C. After mixing, the beads were washed three times with binding buffer and then washed twice with IP buffer [200 mM KCl, 5 mM MgSO4, 2 mM EGTA, 0.5 mM dithiothreitol, and 20 mM Hepes (pH 7.8)] containing 3% BSA. The dynein fraction from sucrose density gradients was dialyzed against IP buffer overnight at 4°C and diluted 1:2 with IP buffer containing 3% BSA. The dynein fraction was added to the beads and mixed on a rotator for 1 hour at 4°C. The beads were washed three times with IP buffer without BSA, suspended in SDS sample buffer, and incubated at 95°C for 2 min. The SDS-treated samples were subjected to SDS-PAGE. Immunoprecipitates were detected by Western blotting using anti–Ci-IC116, anti–Ci-IC110, anti–Ci-IC105, and anti-DYBLUP antibodies. Nonimmune serum from mice was used as a negative control.

In Chlamydomonas, axonemal proteins from WT and mutant cells were extracted with 0.6 M KCl in HMDEK buffer containing EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Tokyo, Japan). The extracts were diluted 10-fold with IP buffer containing 2% polyvinylpyrrolidone (molecular weight 40,000, PVP40; Sigma-Aldrich), 0.05% Triton X-100, and HMDEK and added to rProteinG Sepharose beads (GE Healthcare, Chicago, IL, USA) conjugated with anti–HA-tag polyclonal antibody and incubated for 1 hour at 4°C in the IP buffer. The beads were washed three times with IP buffer without PVP40 and incubated with SDS sample buffer for 5 min at 95°C. The precipitants were separated by SDS-PAGE and stained with silver, and selected bands were identified by peptide mass fingerprinting analysis by MALDI-TOF/MS (BioGARAGE, Leave a Nest Co. Ltd., Japan).

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