Figure 3

Nek4 is localized at the base of the primary cilia. (A) Nek4 colocalizes with Acetylated tubulin at the base of the cilium. RPE-hTERT cells were seeded on coverslips at high confluence and after 20 h of serum starvation were iced-cold methanol fixed. RPE-hTERT cells showed primary cilia induction which can be observed by mouse anti-Acetylated tubulin (a primary cilia marker) stain, and Nek4 colocalizes with acetylated tubulin at the base of the cilium (arrowhead). The nucleus was stained with DAPI. The images were acquired in HC PL APO CS2 63x/1.40 with OIL objective. (B) Nek4 shows a similar localization pattern as its putative interactor Whirlin. HEK293 cells were seeded on Cell Carrier 384 well plates coated with poly-L-lysine (Perkin Elmer), incubated for two days at 37°C with 5% CO2, then fixed with methanol and stained with proper antibodies. The nuclei were stained with Hoechst. Images were acquired with the Operetta automated microscope (Perkin Elmer) in non-confocal mode using the 60x high NA objective (NA = 0.9). The datasets were imported into Volocity for colocalization analysis and contrast correction. The images are representative of at least 10 replicate experiments.