(A) Schematic representation of the domain structure and reported serine phosphorylation sites on the CCDC8 protein. The sequences adjacent to the putative phosphorylated serines and their predicted kinases are shown. (B) U2OS and HEK293 cells were treated with kinase inhibitors targeting GSK3 (by CHIR-98012), CK2 (by CX4945), CDK1 (by RO-3306), and PKC (by sotrastaurin), and lysates were separated by either 10% conventional SDS-PAGE or 6% Zn Phos-tag PAGE, followed by Western blotting using an antibody specific for CCDC8. (C) U2OS cells with endogenous CCDC8 3×FLAG-tagged by CRISPR were treated with GSK3 or CK2 inhibitors. Endogenous CCDC8 was precipitated by anti-FLAG antibody, followed by Western blot with anti-CCDC8, anti–phosphorylated serine 142 (α–p-Ser142), or anti–phosphorylated serine 146 (α–p-Ser146) antibodies. (D) Purified recombinant CCDC8 proteins were incubated with or without recombinant CK2 in kinase buffer containing ATP-γ-S, followed by treatment with p-nitrobenzyl mesylate to alkylate thiophosphates and form thiophosphate ester epitopes. In vitro phosphorylation of CCDC8 by CK2 was detected by Western blot with an antibody recognizing the thiophosphate ester. (E) Recombinant CCDC8 proteins were immobilized on magnetic beads, incubated first with CK2 in buffers containing ATP, and then with GSK3 in buffers containing ATP-γ-S. The phosphorylation by GSK3 was detected by alkylation with p-nitrobenzyl mesylate and Western blot with an antibody recognizing the thiophosphate ester.