Figure 2
From: VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle
Cajal bodies are rescued by wild-type VRK1 but not by kinase-dead VRK1.
A. Rescue of defective Cajal Body formation by murine VRK1. HeLa cells were transfected with siVRK1-02 to knock down the endogenous human VRK1, or with siControl. Cells were retransfected with plasmid expressing murine VRK1-myc-DKK, or with the same plasmid containing the K179E substitution that is kinase-dead. After retransfection cells were immunostained with anti-myc polyclonal antibody to identify those cells expressing exogenous VRK1 active or kinase-dead. Cajal Bodies were visualized by staining with anti-Coilin monoclonal antibody Pdelta (SantaCruz). Quantification of the number of Cajal Bodies (graphs) in the rescue experiment were determined using ImageJ software (NIH). Means of the number (percentage) of cells with or without Cajal Bodies and standard deviations are represented in the graph. The number of analysed cells is indicated at the bottom. *(P < 0.05) **(P < 0.005) ***(P < 0.0005). Efficiency of endogenous VRK1 silencing and expression of murine VRK1 (mVRK1), active or kinase-dead (K179E) were determined by Western blot. B. Murine VRK1 that is active, but not its kinase-dead mutant (K179E), phosphorylated coilin in Ser184. Two cell lines, HEK293T (left) and HeLa (right) were transfected with plasmid expressing active murine VRK1, or inactive murine VRK1(K179E), both tagged with a myc epitope. These tagged murine VRK1 were immunoprecipitated and used for an in vitro kinase assay with coilin-myc as substrate. Phosphorylation of coilin was detected with a specific antibody for Ser184-P.
