Fig. 1: VRK2 directly interacts with G3BP1 and phosphorylates its Ser149 residue.

A Representative immunoblot of pNTAP-streptavidin pull down assay performed with pNTAP-Mock or pNTAP-VRK2 transfected U2OS cells. B Representative immunoblot of GST pull down assay performed with recombinant GST-G3BP1 and His-VRK2. C Autoradiograph (γ-³²P) and Coomassie brilliant blue (CBB) staining from in vitro kinase assay using recombinant GST-G3BP1 and His-VRK2; WT: wild type, S149A: mutation of Ser149 to alanine. Representative immunoblot (D) and quantification (E) of VRK2 knockdown experiments. U2OS cells were transfected with small interfering RNA for control (siCon) or for VRK2 (siVRK2). The phosphorylation of G3BP1 is normalized to total G3BP1, and GAPDH was used as loading control (n = 3). Representative immunoblot (F) and quantification (G) of VRK2 overexpression experiments. U2OS cells were transfected with Flag-Mock, Flag-VRK2, or Flag-VRK2 kinase dead form (VRK2 KD). The phosphorylation of G3BP1 is normalized to total G3BP1 and GAPDH was used as loading control (n = 3). n.s., not significant, *** p ≤ 0.001; unpaired Student’s t test was performed for (E); ordinary one-way ANOVA with Tukey’s multiple comparison test was performed for (G). The “n” represents the number of independent experiments. Error bars indicate SDs.