Fig. 4: VRK2 is downregulated by RNF144A through proteasomal degradation.

A mRNA level of VRK2 was measured in stress-induced U2OS cells. U2OS cells were treated with SA to induce stress (50 μM, 8 h). mRNA level of GAPDH was used for normalization (n = 3). Representative immunoblot (B) and the quantification (C) of VRK2 under stress and inhibition of proteasomal degradation. Stress was induced in U2OS cells with SA (50 μM, 8 h) and proteasomal degradation was inhibited using MG-132 (20 μM, 8 h). GAPDH was used for normalization (n = 3). Representative immunoblot (D) and the quantification of VRK2 protein level (E) and VRK2 mRNA level (F) in RNF144A-excessive U2OS cells. U2OS cells were transfected with GFP-RNF144A and were incubated for 24 h. Protein level of GAPDH (n = 5) and mRNA level of GAPDH (n = 3) was used for normalization. Representative immunoblot (G) and the quantification of VRK2 protein level (H) and VRK2 mRNA level (I) in RNF144A-deficient U2OS cells. U2OS cells were transfected with siRNF144A and were incubated for 24 h. Protein level of GAPDH (n = 3) and mRNA level of GAPDH (n = 3) were used for normalization. J Representative immunoblot of pNTAP-streptavidin pull down assay performed with pNTAP-Mock or pNTAP-VRK2 transfected U2OS cells. GAPDH was used for normalization. K Representative immunoblot of GST pull down assay performed with recombinant GST-RNF144A and Intein-VRK2. L Representative immunoblot of poly-ubiquitination assay. GAPDH was used for normalization. n.s., not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; unpaired Student’s t test was performed for (A, C, E, F, H, I). The “n” represents the number of independent experiments. Error bars indicate SDs.