Nek4 copurifies with the mitochondrial fraction. (A) Nek4 is present in mitochondrial fractions and shows a similar perinuclear pattern of localization (B) such as its putative interactor ANT3, a mitochondrial protein. HEK293 cells were seeded on Cell Carrier 384 well plates coated with polylysine (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. The mitochondrial localization of Nek4 was verified by cell fractioning of HEK293 Flp-In (stably expressing Nek4 isoforms or empty vector - Flag). Using Qproteome Mitochondria Isolation Kit (QIAGEN). N: nuclear, M: mitochondrial and C: cytosolic fractions.