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. 2017 Dec 19;6:e29953. doi: 10.7554/eLife.29953

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© 2017, Wakida et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A) The RAD9 C-terminus co-precipitates with PLK1. GST-RAD9 (3A: S277A, S328A, S336G) (30 pmol) was pre-incubated with CDK2-Cyclin A2 (0.5 pmol) in 15 µl of kinase buffer at 30 ˚C for 30 min, followed by an incubation with 4 pmol of PLK1 (30 ˚C for 5 min followed by 4 ˚C for 30 min). The reaction mixture was captured with GSH-beads for 30 min at 4 ˚C. A schematic of the experiment is shown (left). A western blot analysis was performed using α-PLK1 (PLK1) and α-pT292 (pT292) antibodies, and a silver-stained gel is also shown (right). (B) CDK and Thr292 of RAD9 are responsible for the PLK1-RAD9 interaction. A GSH-bead pull down assay was performed as described in (A), except that the reactions without CDK2-CyclinA2 or GST-RAD9-T292A (T292A) mutant protein were added to the experiment. A western blot analysis was performed using α-PLK1 and α-RAD9 antibodies. (C) The Thr292-phosphorylated peptide can compete with the RAD9-PLK1 interaction. PLK1 (4 pmol) was pre-incubated with phospho- or non-phospho-peptides (8 nmol or 0.8 nmol, Non-P: non-phosphorylated peptide, pT292: a peptide phosphorylated on Thr292, pS291: a peptide phosphorylated on Ser291, pS291/pT292: a peptide phosphorylated on both Ser291 and Thr292; for the pS291/pT292 peptide, only 8 nmol was tested) for 30 min at 4 ˚C, and then mixed with GST-RAD9A (3A) (30 pmol), which was phosphorylated by CDK2-CyclinA2 (0.5 pmol) for 30 min at 30 ˚C in a 20 µl reaction. The reaction mixture was incubated with GSH-beads for 20 min at 4 ˚C. A schematic drawing of the experiment is shown (left). A western blot analysis was performed using α-PLK1, and a silver stained gel is also shown (right). (D) The T292 phosphorylated peptide can promote PLK1-dependent phosphorylation of RAD9. GST-RAD9-T292A (3A) (10 pmol) was incubated with PLK1 (0.3 pmol) and non-phospho- or phospho-peptides (0.8 nmol: same peptides used in (C)), in a 20 µl reaction for 20 min at 30 ˚C. A schematic drawing of the experiment is shown (left). A western blot using the α-RAD9 antibody is also shown (right). (E), (F) RAD9 co-immunoprecipitates with PLK1. Lysates prepared from 293A-T-REx cells stably expressing WT or T292A-mutated RAD9-mH (RAD9-S391A/T292A expressing cells were used for T292A) were subjected to immunoprecipitation, using α-myc antibody-coated agarose beads. Western blots were performed using α-RAD9 and α-PLK1 antibodies on the input (Input) and immunoprecipitates (IP). Ectopically expressed PLK1-HA was used in (E) and endogenously expressed PLK1 was detected in (F). See also Figure 2—figure supplement 1. PLK1 interacts with and phosphorylates RAD9.

Figure 2—figure supplement 1. — (A) The RAD9 C-terminus co-precipitates with PLK1. GST-RAD9 (3A: S277A, S328A, S336G) (30 pmol) was pre-incubated with CDK2-Cyclin A2 (0.5 pmol) in 15 µl of kinase buffer at 30 ˚C for 30 min, followed by an incubation with 4 pmol of PLK1 (30 ˚C for 5 min followed by 4 ˚C for 30 min). The reaction mixture was captured with GSH-beads for 30 min at 4 ˚C. A schematic of the experiment is shown (left). A western blot analysis was performed using α-PLK1 (PLK1) and α-pT292 (pT292) antibodies, and a silver-stained gel is also shown (right). (B) CDK and Thr292 of RAD9 are responsible for the PLK1-RAD9 interaction. A GSH-bead pull down assay was performed as described in (A), except that the reactions without CDK2-CyclinA2 or GST-RAD9-T292A (T292A) mutant protein were added to the experiment. A western blot analysis was performed using α-PLK1 and α-RAD9 antibodies. (C) The Thr292-phosphorylated peptide can compete with the RAD9-PLK1 interaction. PLK1 (4 pmol) was pre-incubated with phospho- or non-phospho-peptides (8 nmol or 0.8 nmol, Non-P: non-phosphorylated peptide, pT292: a peptide phosphorylated on Thr292, pS291: a peptide phosphorylated on Ser291, pS291/pT292: a peptide phosphorylated on both Ser291 and Thr292; for the pS291/pT292 peptide, only 8 nmol was tested) for 30 min at 4 ˚C, and then mixed with GST-RAD9A (3A) (30 pmol), which was phosphorylated by CDK2-CyclinA2 (0.5 pmol) for 30 min at 30 ˚C in a 20 µl reaction. The reaction mixture was incubated with GSH-beads for 20 min at 4 ˚C. A schematic drawing of the experiment is shown (left). A western blot analysis was performed using α-PLK1, and a silver stained gel is also shown (right). (D) The T292 phosphorylated peptide can promote PLK1-dependent phosphorylation of RAD9. GST-RAD9-T292A (3A) (10 pmol) was incubated with PLK1 (0.3 pmol) and non-phospho- or phospho-peptides (0.8 nmol: same peptides used in (C)), in a 20 µl reaction for 20 min at 30 ˚C. A schematic drawing of the experiment is shown (left). A western blot using the α-RAD9 antibody is also shown (right). (E), (F) RAD9 co-immunoprecipitates with PLK1. Lysates prepared from 293A-T-REx cells stably expressing WT or T292A-mutated RAD9-mH (RAD9-S391A/T292A expressing cells were used for T292A) were subjected to immunoprecipitation, using α-myc antibody-coated agarose beads. Western blots were performed using α-RAD9 and α-PLK1 antibodies on the input (Input) and immunoprecipitates (IP). Ectopically expressed PLK1-HA was used in (E) and endogenously expressed PLK1 was detected in (F). See also Figure 2—figure supplement 1. PLK1 interacts with and phosphorylates RAD9.