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. 2020 Dec 22;220(1):e202002151. doi: 10.1083/jcb.202002151

Figure 5.

Figure 5.

PLK4 phosphorylation of NEDD1 regulates its interaction with SAS-6 for cartwheel assembly. (A) U2OS cells were treated with either DMSO or 500 nM centrinone B for 24 h and coimmunostained with antibodies against NEDD1 and SAS-6. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (B) Total extracts of HEK293T cells treated with DMSO or 500 nM centrinone B for 24 h were immunoprecipitated with anti–SAS-6 antibody and probed with antibodies against SAS-6 and NEDD1. (C) Total extracts of HEK293T cells transfected with GFP-tagged WT or 6A mutant NEDD1 were immunoprecipitated with GFP-Trap beads and probed with antibodies against SAS-6, γ-tubulin, and GFP. (D) Total extracts of HEK293T cells transfected with GFP-tagged WT or S325A mutant NEDD1 were immunoprecipitated with GFP-Trap beads and probed with antibodies against SAS-6, γ-tubulin, and GFP. (E) Partial protein sequences of NEDD1 in human, mouse, rat, horse, monkey, bovine, zebrafish, xenopus, and chicken. (F) U2OS cells were cotransfected with endogenous NEDD1 siRNA and Myc-tagged siRNA-resistant WT or S325A mutant NEDD1 and immunostained with anti-SAS-6 antibody. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (G) Comparisons of the relative fluorescence intensity of SAS-6 in (F). Three independent replicates of >30 cells per replicate were quantified. (H) U2OS cells were cotransfected with endogenous NEDD1 siRNA and Myc-tagged siRNA-resistant WT or S325A mutant NEDD1 and coimmunostained with antibodies against Myc and Centrin1. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (I) Percentages of the cells with different numbers of centrioles and comparisons of those cells with fewer than four centrioles in each treatment in H. Three independent replicates of >100 cells per replicate were quantified. (J) Total extracts of HEK293T cells transfected with GFP-tagged WT or S325A mutant NEDD1 were immunoprecipitated with GFP-Trap beads and probed with antibodies against γ-tubulin, GCP2, GCP3, GCP4, GCP5, GCP6, and GFP. Error bars in G and I indicate mean ± SD. ***, P < 0.001 (two-tailed t test). Tub, tubulin.