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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Cancer Lett. 2021 Nov 20;526:53–65. doi: 10.1016/j.canlet.2021.11.018

Fig. 2. CTDSPL2 is phosphorylated at T86, S104, and S134 by CDK1.

Fig. 2.

(A) HEK293T cells were transfected with Flag–CTDSPL2 and treated with nocodazole or Taxol for 16 h. Indicated phospho-sites were tested in total cell lysates and immunoprecipitated (IP) samples. (B, C) S2.013 cells were treated with nocodazole or Taxol for 24 h. Lysates were electrophoresed on SDS polyacrylamide gels with non-phospho-peptide or specific phospho-peptide blocking and probed with phospho-antibodies. (D, E) Control and CTDSPL2-knockdown PANC-1 (D) and S2.013 (E) cells were subjected to Taxol treatment for 24 h. Total cell lysates were probed with the indicated phospho-antibodies. (F) HEK293T cells were transfected with CTDSPL2-WT or CTDSPL2–4A plasmid. Total cell lysates were probed with the indicated phospho-antibodies. WT: wild type. 4A: T86A/S104A/S134A/S165A. (G) HeLa cells were treated with Taxol alone or together with RO3306 or Purvalanol A and lysates were harvested for western blotting analysis. Increased phospho-T320 PP1α and Cyclin B1 levels mark the cells in mitosis. PP1α (p-T320) is a well-known substrate of CDK1 and used to measure the CDK1 activity. (H) HEK293T cells were transfected with indicated expression constructs and total cell lysates were analyzed by western blotting. GFP-Cyc B1-CA: GFP-Cyclin B1-R42A (a nondegradable/constitutive active mutant). Flag-CDK1-CA: Flag-CDK1-T14A/Y15A (non-phosphorylatable/constitutive active CDK1). (I) TetOn inducible CDK1 knockdown HeLa cells were treated as indicated and lysates were harvested for western blotting analysis.