Fig. 5: CK2α interacts with DUB3 and phosphorylates DUB3 at Thr495.

A The direct interaction between bacterial purified GST-CK2α and His-DUB3 was measured by an in vitro interaction assay. CBS was performed to examine GST or GST-fusion protein levels. Cell lysates of A2780 and SKOV3 were immunoprecipitated using IgG, anti-CK2α (B), or anti-DUB3 (C) antibodies. The endogenous interaction of CK2α-DUB3 was measured by immunoblotting. D Vector (V) or Flag-DUB3 were transfected into A2780 cells. Cell lysates were immunoprecipitated using anti-Flag affinity gel, and the phosphorylation of DUB3 was examined by phospho-CK2 substrate antibody. E Indicated plasmids were transfected into cells and treated with or without Silmitasertib for 2 h. Anti-Flag affinity gel was used to immunoprecipitate Flag-DUB3, and a phospho-CK2 substrate antibody was used to measure DUB3 phosphorylation by immunoblotting. F Indicated constructs were transfected into cells. HA-protein agarose was used to immunoprecipitate DUB3, and a phospho-CK2 substrate antibody was used to measure the phosphorylation of DUB3. G Bacterial purified GST, GST-DUB3 or GST-DUB3 T495A were incubated with immunoprecipitated HA-CK2α by HA-protein agarose in the kinase reaction buffer. A Phospho-CK2 substrate antibody was used to measure the phosphorylation of DUB3. CBS was performed to examine GST and GST-fusion protein levels. H In vitro kinase assay was performed by mixing purified WT GST-CK2α or GST-CK2α K68M proteins with purified His-DUB3 WT or His-DUB3 T495A proteins. A phospho-CK2 substrate antibody was used to detect the phosphorylation levels of DUB3. I Indicated constructs were transfected into cells. HA-protein agarose was used to immunoprecipitate DUB3, and a phospho-CK2 substrate antibody was used to measure the phosphorylation of DUB3.