The PLK1 PBD binds to the Thr887-Ser888-Thr889 motif on MLL5. (A) The central domain of MLL5 binding to HA-PLK1. 293T cells expressing FLAG-MLL5 truncated mutant and HA-PLK1 were synchronized to prometaphase by nocodazole treatment. The cell lysates were immunoprecipitated with anti-HA antibody or mouse IgG and detected by Western blotting (WB) with anti-FLAG and anti-HA antibodies. Arrowhead indicates the slower-migrating form of FLAG-MLL5-CD. PS, PHD/SET domain; CT, C-terminal domain. (B) Loss of interaction between PBD-mutated PLK1 and the slowest-migrating form of FLAG-MLL5-CD. 293T cells expressing FLAG-MLL5-CD and HA-PLK1 or its mutants were synchronized to prometaphase. The cell lysates were immunoprecipitated with anti-HA antibody and detected by Western blotting with anti-FLAG and anti-HA antibodies. Arrowhead indicates the faster-migrating form of MLL5-CD; arrow indicates the slower-migrating form of MLL5-CD with PTMs. KD*, kinase dead; PBD*, PBD mutated. (C and D) Reduced interaction between PLK1 and the slowest-migrating form of TST887AAA, T887A, S888A, or T889A mutant. 293T cells expressing FLAG-MLL5-CD4 or its mutants and HA-PLK1 were synchronized to prometaphase before immunoprecipitation and Western blotting. Arrow indicates the slowest-migrating form of MLL5-CD4 or its mutant; * indicates the interaction between TST887AAA mutant and PLK1. Results are representative of at least three experimental repeats.