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Fig. 1. Cnk2p-HA is an axonemal protein. (A) Schematic representation of the CNK2-HA expression construct, containing the CNK2 coding sequence (CDS) with the 3XHA epitope tag on the C-terminus. The PsaD promoter and UTRs are used to drive constitutive expression (Fischer and Rochaix, 2001). E, EcoRI; H, HindIII; N, NdeI restriction sites. (B) Immunoblot analysis of cell fractions from Cnk2p-HA expressing cells probed with an anti-HA antibody. Blots were stripped and reprobed with an anti- ${alpha}$ -tubulin antibody. Cell equivalent amounts of whole cells (WC), cell bodies (CB) and flagella (Flag) were loaded in each lane (left-hand blot). Stoichiometric amounts of flagella (Flag), membrane and matrix fraction (M+M), and axonemal fractions (Axo) were loaded in each lane (right-hand blot). (C) Wild type and two examples of Cnk2p-HA cells triple-stained for indirect immunofluorescence with anti- ${alpha}$ -tubulin, anti-HA and anti-centrin antibodies. Bar, 5 µm.

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