Fig. 4.

Subcellular localization of endogenous VRK1 and Ran proteins and detection of their interaction by reciprocal immunoprecipitations. A, confocal immunofluorescence microscopy of endogenous VRK1 and Ran proteins in HEK293T cells. VRK1 was detected with the VE1 polyclonal antibody, and as secondary antibody a Cy3-labeled anti-rabbit antibody was used. Ran was detected with a monoclonal antibody, and as secondary antibody a Cy2-labeled anti-mouse antibody was used. B, colocalization of endogenous Ran and VRK1 in cells that have completed division. C, HEK293T cells (2 × 10⁸) were lysed, and the extracts were subjected to immunoblot analysis to check the expression of endogenous Ran and VRK1 (top panel) with specific antibodies. Half of the extracts were used to perform an immunoprecipitation (IP) of endogenous Ran with the specific monoclonal antibody and a control immunoprecipitation with a nonspecific monoclonal antibody (αAU5) (left panel). The other half of the extracts was used to immunoprecipitate endogenous VRK1 with a mixture of two specific polyclonal antibodies (VE1 and VC1) and to perform a control immunoprecipitation with a nonspecific polyclonal antibody (αFLAG) (right panel). The immunoprecipitations were subjected to immunoblot (IB) analysis. VRK1 was detected in the Ran immunoprecipitation but not in the negative control, and Ran was detected in the VRK1 immunoprecipitation but not in the negative control. D, as a positive control, the same conditions that were used to detect the interaction of VRK1 with immunoprecipitated Ran were used to see the interaction of RCC1 with immunoprecipitated Ran. E, colocalization of endogenous Ran and VRK2 in HEK293T cells. VRK2 protein was detected with a rabbit polyclonal antibody. Ran was detected as described above. DAPI, 4′,6-diamidino-2-phenylindole.