Nek5 localizes to the centrosome. (a) RT-PCR of Nek5 and actin with RNA from U2OS or Hek293 cells. (b) RT-PCR of Nek5 and GAPDH with RNA from cancer cell lines (blue, colorectal; black, breast; orange, pancreatic) and immortalized breast epithelial cells (HBL100; bold). (c) Western blot analysis of U2OS cells that were mock-transfected or transfected with GFP-Nek5 for 24 h. Blots were probed with the in-house Nek5 antibody. α-Tubulin was used as loading control. (d) Staining of U2OS cells with either an in-house or commercial Nek5 antibody and γ-tubulin. (e) Staining of Nek5 (with in-house antibody) and γ-tubulin in U2OS cells transfected with Nek5- or GL2-specific siRNAs for 48 h. (f) GFP-Nek5–transfected U2OS cell stained for γ-tubulin. (g) HeLa::centrin1-GFP cell stained for Nek5. (h) U2OS cells stained for Nek2, rootletin, or C-Nap1 and Nek5. The schematic illustrates relative localization of proteins within centrioles. (i) Staining of a ciliated hTERT-RPE-1 cell for Nek5 and acetylated tubulin. (j) Staining of γ-tubulin and Nek5 in U2OS cells at the stages indicated. The intensity of Nek5 at the centrosome relative to interphase is indicated. Data are mean ± SD of at least 20 centrosomes (n = 2). Bars: (d–g and i–j, main images) 10 µm; (d–f, g, and i–j, insets, and h) 1 µm.