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. 2008 Nov;7(11):2199–2214. doi: 10.1074/mcp.M700586-MCP200

Fig. 5.

Fig. 5.

Mapping the VRK1 region that interacts with Ran. A, mapping the interaction between GST-Ran and VRK1. HEK293T cells were transfected with constructs expressing the full-length VRK1 (plasmid pCEFL-VRK1-HA), amino-terminal region 1–332 (pcDNA-VRK1-N-myc), carboxyl-terminal region 267–396 (pEGFP-C-VRK1), or the kinase-dead VRK1 (pCDNA-VRK1-K179E-myc). The lysates were incubated with 10 μg of GST-Ran purified protein and centrifuged, and the proteins in the pulldown were identified with a specific polyclonal antibody for VRK1 (lower panel). The expression of the proteins was determined by Western blot (top panel). B, diagram illustrating the common region between the different VRK1 constructs and identifying the region of VRK1 needed for interaction with Ran. C, pulldown of endogenous Ran with different constructs of human VRK2. HEK293T cells were transfected with plasmids pCEFL-GST, pCEFL-GST-VRK2N (1–320), pCEFL-GST-VRK2A-C1 (256–508), pCEFL-GST-VRK2A-C2 (364–508), and pCEFL-GST-VRK2B-C (256–397) expressing different regions of human VRK2. In the pulldown proteins the presence of endogenous Ran was determined with a specific antibody. D, diagram illustrating the common region between the different VRK2 constructs and identifying the region of VRK2 needed for interaction with Ran. E, interaction of RanL43E with VRK1. The experiment was performed as in A. F, partial competition of VRK3 with VRK1 for binding to Ran. The mixture of proteins in the concentrations indicated in the figure was used for a pulldown of His-VRK1, and the bound proteins were determined by immunoblot (IB). wt, wild type.