Figure 4.
Figure 4. CEP164 binds and recruits Cby to the distal appendages. (A) Schematic diagram of CEP164 constructs used in this study. The numbers indicate amino acid positions. (B) HEK293T cells were transfected with HA-CEP164 and Flag-Cby expression constructs as indicated, and cell lysates were immunoprecipitated with the Flag antibody and immunoblotted with the HA antibody. Cell lysates were also probed with HA and Flag antibodies to show stable protein expression. (C) Cell lysates from HEK293T cells expressing Flag-Cby and the indicated HA-tagged CEP164 fragments were immunoprecipitated with the Flag antibody and detected with the HA antibody. del., deletion. (D) CEP164 binds to the C-terminal region of Cby. HEK293T cells were transfected with expression constructs for HA–CEP164-CC and Flag-tagged Cby-FL, -4A, or -C (aa 64–126), and cell lysates were immunoprecipitated with the Flag antibody and immunoblotted with the HA antibody. (right) The N-terminal half of Cby (aa 1–63) was undetectable upon transfection. Cells were cotransfected with an expression plasmid for Flag-EGFP to normalize for transfection efficiency. White lines indicate that intervening lanes have been spliced out. (E) HeLa cells expressing GFP–centrin-1 were mock treated (all reagents except for siRNA) or transfected with CEP164 siRNA and immunostained with CEP164 and Cby antibodies. Cells with a pair or two pairs of centrioles are shown for each siRNA. Bar, 1 µm. (F) Quantification of the number of cells with Cby at the mother centrioles after siRNA treatment. U2OS cells were untreated, mock transfected, or transfected with CEP164 siRNA for 27 h and immunostained for Cby and CEP164. A total of 811 (untreated), 595 (mock), and 856 (CEP164 siRNA) cells were counted from at least five independent experiments. Data are presented as means ± SD. ****, P < 0.0001. Expression levels of CEP164, Cby, and GAPDH were determined by Western blotting. The asterisk indicates nonspecific bands. The band intensities of CEP164 and Cby were quantified and normalized to those of GAPDH. The normalized values for mock-transfected samples were set as 1. IB, immunoblot; IP, immunoprecipitation.

CEP164 binds and recruits Cby to the distal appendages. (A) Schematic diagram of CEP164 constructs used in this study. The numbers indicate amino acid positions. (B) HEK293T cells were transfected with HA-CEP164 and Flag-Cby expression constructs as indicated, and cell lysates were immunoprecipitated with the Flag antibody and immunoblotted with the HA antibody. Cell lysates were also probed with HA and Flag antibodies to show stable protein expression. (C) Cell lysates from HEK293T cells expressing Flag-Cby and the indicated HA-tagged CEP164 fragments were immunoprecipitated with the Flag antibody and detected with the HA antibody. del., deletion. (D) CEP164 binds to the C-terminal region of Cby. HEK293T cells were transfected with expression constructs for HA–CEP164-CC and Flag-tagged Cby-FL, -4A, or -C (aa 64–126), and cell lysates were immunoprecipitated with the Flag antibody and immunoblotted with the HA antibody. (right) The N-terminal half of Cby (aa 1–63) was undetectable upon transfection. Cells were cotransfected with an expression plasmid for Flag-EGFP to normalize for transfection efficiency. White lines indicate that intervening lanes have been spliced out. (E) HeLa cells expressing GFP–centrin-1 were mock treated (all reagents except for siRNA) or transfected with CEP164 siRNA and immunostained with CEP164 and Cby antibodies. Cells with a pair or two pairs of centrioles are shown for each siRNA. Bar, 1 µm. (F) Quantification of the number of cells with Cby at the mother centrioles after siRNA treatment. U2OS cells were untreated, mock transfected, or transfected with CEP164 siRNA for 27 h and immunostained for Cby and CEP164. A total of 811 (untreated), 595 (mock), and 856 (CEP164 siRNA) cells were counted from at least five independent experiments. Data are presented as means ± SD. ****, P < 0.0001. Expression levels of CEP164, Cby, and GAPDH were determined by Western blotting. The asterisk indicates nonspecific bands. The band intensities of CEP164 and Cby were quantified and normalized to those of GAPDH. The normalized values for mock-transfected samples were set as 1. IB, immunoblot; IP, immunoprecipitation.

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