Figure 5. Functional interaction of Cep152 and Plk4. (A) Plk4 is lost from the centrosome in cells expressing GFP-Cep152 (1–217). U2OS cells transfected with GFP or GFP-Cep152 (1–217) were fixed 24 h after transfection and labeled with antibodies to GFP, Plk4, and γ-tubulin. Numbered boxes identify enlarged images shown on right. Bars (boxes) 1 µm; (fields) 5 µm. (B) U2OS cells were depleted of Plk4 or Cep152 by siRNA transfection, fixed 72 h after transfection, and labeled with antibodies to Cep152, Plk4, and γ-tubulin. Image pairs represent centrosomes in cells from the same coverslip taken with the same camera settings. (C) U2OS cells transfected for 72 h with either control siRNA (Ctl) or Cep152 siRNAs were arrested in S phase by incubation in aphidicolin for 24 h and fixed and labeled with antibodies to centrin, Sas6, and Cep152. 25% of Cep152-depleted cells had reduced Sas6 labeling on both centrioles (middle), 65% had no Sas6 labeling (bottom), and 10% had centrioles that differed in the amount of Sas6 (n = 150 cells). Exposure and gain conditions were constant for Sas6 image acquisition. (B and C) Bars, 1 µm. (D) Cep152 can be phosphorylated by Plk4 in vitro. Purified GST-Plk4, either wild type (WT) or kinase dead (KD), was incubated in the presence of [32P]ATP (32P) with full-length GFP-Cep152 (left) or GFP-Cep152 (1–217; middle) isolated from HEK293T cells by anti-GFP immunoprecipitation (IP) or with MBP-Cep152 (1–217; right) purified from E. coli. Proteins were visualized by Western blotting (Ab) of 0.1× kinase assay input using antibodies to GFP (left and middle) or by Coomassie stain (C) of the gel (right). Bands marked with an asterisk indicate autophosphorylated Plk4. Cep152 32P incorporation was normalized within each gel group. Black lines indicate that gel lanes imaged by different methods were spliced together.
Image published in: Hatch EM et al. (2010)
© 2010 Hatch et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license
Permanent Image Page
Printer Friendly View
XB-IMG-125496