Fig. 4.

Loss of trypanin weakens the association of CMF70 with the axoneme. (A) Protein fractions, as defined in Fig. 2, were prepared from CMF70-HA cells harboring a Tet-inducible trypanin RNAi knockdown construct grown with (+) or without (−) 1 μg/ml tetracycline for 72 hours. Samples were analyzed by SDS-PAGE and immunoblotting with antibodies against trypanin (TPN, top panel) or HA (CMF70-HA, middle panel), or stained for total protein with Sypro-ruby (lower panel). (B) P2 flagellar skeletons from uninduced (−) and Tet-induced (+) trypanin RNAi CMF70-HA cells were extracted with the indicated concentration of KI and separated by centrifugation into supernatants (S) and pellets (P). Samples were analyzed as in A. Treatment with 0.3 M KI partially solubilized CMF70-HA from the axoneme when trypanin was ablated by RNAi but not in control cells. Results are representative of three independent experiments.