Fig. 7.

Interdependence between FAZ27 and ClpGM6 for assembly into the FAZ. (A) Effect of FAZ27 RNAi on the localization of ClpGM6 to the flagellum. ClpGM6 was tagged with an N-terminal triple HA epitope in the FAZ27 RNAi cell line. Cells were co-immunostained with FITC-conjugated anti-HA antibody and anti-CC2D antibody. Cells were counterstained with DAPI to stain nuclear and kinetoplast DNA. The white brackets indicate the regions without 3HA–ClpGM6 signal. Scale bar: 5 μm. (B) Effect of FAZ27 RNAi on the distribution of 3HA–ClpGM6 in cytosolic and cytoskeletal fractions. The soluble cytosolic (S) fraction and cytoskeletal pellet (P) fraction were prepared as described in Fig. 6B. 3HA–ClpGM6 was detected by anti-HA antibody. α-tubulin and TbPSA6 served as cytoskeleton and cytosol markers, respectively. (C) Effect of ClpGM6 depletion on FAZ27 localization. FAZ27 was endogenously tagged with a triple HA epitope in the FLAM3 RNAi cell line. Cells were immunostained with FITC-conjugated anti-HA antibody to detect FAZ27-3HA and anti-CC2D antibody to label the FAZ. Cells were counterstained with DAPI to stain nuclear and kinetoplast DNA. The white brackets indicate the regions without FAZ27–3HA signal. Arrowheads indicate the FAZ27 protein located at the proximal end of the new FAZ. Scale bar: 5 μm. (D) Effect of ClpGM6 RNAi on the distribution of FAZ27 in cytosolic and cytoskeletal fractions. The soluble cytosolic (S) fraction and cytoskeletal pellet (P) fraction were prepared as described in Fig. 6B. FAZ27–3HA was detected by anti-HA antibody. α-tubulin and TbPSA6 served as cytoskeleton and cytosol markers, respectively. Images in A,C and blots in B,D are representative of three experiments.