CHK2 localizes at centrosomes in mitotic cells but not in interphase. U2OS cells were transduced with lentiviruses coding for GFP-CHK2 or Flag-CHK2 and stably selected. (A) Cells were exposed for 48 h to doxycycline at 5 ng/ml for GFP-CHK2 cells, or 20 ng/ml for Flag-CHK2 cells, and expression of proteins in whole cell extracts was analyzed by Western blotting using the indicated antibodies. The arrows indicate endogenous and exogenous CHK2 proteins. Note that some exogenous CHK2 is expressed without tag. β-actin was used as loading control. (B and C) GFP-CHK2 and Flag-CHK2 are exclusively localized in the nucleus of interphase cells whereas a small subpopulation localizes at the centrosomes during mitosis. 48 h following transgene induction cells were fixed in methanol. Centrosomes were stained with anti-γ-tubulin antibody (red), and DNA was counterstained with DAPI (blue). The localization of GFP-CHK2 was observed by direct fluorescence and Flag-CHK2 was immunostained with an anti-Flag antibody (green). Cells in interphase and various phases of mitosis were selected.