Figure 5

Nek4 interacts and colocalizes with DNA damage response proteins. (A) Cells expressing empty vector (Flag), isoform 1 (Nek4.1 F), isoform 2 (Nek4.2 F) or kinase dead mutant of Nek4.1 were lysed and phosphoproteins present in these cells were purified. (B) Nek4 and PCNA co-localization is UV exposition-dependent. HEK293T cells were or not (control) UV irradiated (60 J/m²). One hour after irradiation, the cells were methanol fixed and indirect immunofluorescence against Nek4 and PCNA was performed. DNA was stained with DAPI. Only irradiated cells showed a strong Nek4 and PCNA colocalization. (C) Dotted Nek4 and PML localization show partially overlapping patterns. The image presented here is a section from the original image to emphasize the PML bodies. Two fields were analyzed and from these 14 cells with positive staining for PML bodies were observed. Arrows show that some dotted staining of Nek4 corresponds to PML bodies. Seven cells showed nuclear dots to Nek4 and PML. In these nuclei 62% of PML bodies showed positive colocalization with Nek4, and, 27% of Nek4 dot like staining showed positive colocalization with PML bodies. Thus, the partial colocalization of Nek4 with PML indicates that most of PML bodies contain Nek4, but, Nek4 is present also in other nuclear structures in addition to PML bodies. Nek4 and PML stained dots were manually counted using FIJI software. The images were acquired in HC PL APO CS2 63x/1.40 with an OIL objective. T: total, FT: flow through, E: eluate.