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. Author manuscript; available in PMC: 2022 Aug 18.
Published in final edited form as: J Am Chem Soc. 2021 Aug 4;143(32):12675–12687. doi: 10.1021/jacs.1c05386

Figure 2|. NBDY is phosphorylated at T40 downstream of cyclin-dependent kinases (CDKs) at the G2/M checkpoint.

Figure 2|

(a) Mitosis-specific NBDY phosphorylation site (red). (b) NBDY phosphorylation at T40 (pT40) after nocodazole treatment identified by LC-MS/MS. Red peaks, y-ions; yellow peaks, b-ions. c) NBDY pT40 antibody validation. Immunopurified samples were prepared from HEK 293T cells transiently expressing WT or T40A FLAG-NBDY with or without nocodazole treatment. λPPase indicates treatment with λ-phosphatase before SDS–PAGE. (d) Phos-tag PAGE and western blotting validation of NBDY pT40. Immunopurified samples were prepared from HEK 293T cells transiently expressing FLAG-NBDY with or without nocodazole treatment. β-Actin, loading control. (e) Phosphorylation of NBDY is regulated by CDKs. Cells expressing FLAG-NBDY were treated with nocodazole in the presence of a CDKs inhibitor (AT7519) or a PI3K inhibitor (wortmannin) as a negative control before IP and western blotting. (f) Timing of NBDY phosphorylation at T40 downstream of CDKs at the G2/M checkpoint of the cell cycle.