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. 2019 Nov 4;9:15930. doi: 10.1038/s41598-019-50702-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Differential engagement of PLK1 PBD substrates via the Tyr pocket. (A) FRAP analysis of interphase centrosomes in live cells. GFP-PLK1_Wt/AAD/AM at the centrosome was photobleached and its recovery monitored at 0.2 s intervals. The median t_1/2 time taken for 50% maximal recovery for GFP-PLK1_Wt/AAD/AM were found to be 0.7 s, 0.4 s and 0.3 s respectively. The arrowhead indicates the point of photobleaching. An inset shows data points for the first 5 seconds including photobleaching and recovery time of GFP signal. (B) HeLa cells expressing GFP-PLK1_Wt/AAD/AM were synchronized in mitosis by double thymidine block and released as shown in the experimental schedule. The cell lysates were immunoprecipitated using GFP-Trap® beads to pull down GFP-PLK1_Wt/AAD/AM and analysed by immunoblotting. The data presented is representative of two independent experiments. (C) HeLa cells expressing GFP-PLK1_Wt/AAD/AM were transfected with PBIP1_Wt-V5 or pcDNA™3.1/V5-HisA vector (or V5 vector) and harvested 24 h later. PBIP1_Wt-V5/ V5 was pulled down from the lysates using V5 antibody and immunoprecipitates were analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments. (D) HeLa GFP-PLK1_Wt overexpressing cells were transfected with either PBIP1_Wt/F71A/T78A-V5 or V5 vector and harvested 24 h later. PBIP1_Wt/F71A/T78A-V5 was pulled down from the lysates using V5 antibody and analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments.