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

Figure 1. CDK phosphorylates threonine 292 of RAD9.

(A) Schematic of the aim of this manuscript. (B) The recombinant GST-tagged C-terminal (a.a. 266–391) portion of RAD9 was mixed with the purified active CDK2-CyclinA2 complex. Western blotting was performed using the α-RAD9 antibody and the α-pT292 (pT292) and α-phospho-Ser277 (pS277) RAD9 antibodies. (C) Top: The thymidine block and release experiment was performed on HEK293A-T-REx cell lines stably expressing RAD9-WT-mH or RAD9-S291A/T292A-mH. Doxycyclin (0.5 µg/ml) was added during the second thymidine block. The western blotting analysis of cell lysates obtained at the indicated time points is shown, and α-myc, α-pT292 and α-pS277 antibodies were used. The asterisk (*) shows a nonspecific signal. Bottom: The cell cycle profiles were quantified by a flow cytometer analysis. The G1, S, and G2 phases were quantified via propidium iodide staining, and the M phase cells were quantified using an antibody against the phospho-serine 10 of histone H3. (D) A chromatin fractionation assay (Ohashi et al., 2014; Zou et al., 2002) was performed in HEK293A-T-REx cell lines stably expressing RAD9-WT-mH (WT) or RAD9-S291A/T292A-mH (T292A). Cells were grown in media containing 1.5 mM hydroxyurea for 16 hr (+HU) and then released from the hydroxyurea arrest for 1 hr (Rel.). A western blotting analysis is shown, and α-RAD17, α-myc (RAD9), α-phospho-serine 345 of CHK1 (CHK1-pS345), α-phospho-serine 139 of Histone H2AX (p-H2AX), α-H2AX, α-phospho-threonine 68 of CHK2 (CHK2-pT68), and α-tubulin were used. (E) The flow cytometry analysis was performed with U2OS T-REx cells stably expressing RAD9-WT-mH (WT) or RAD9-S291A/T292A-mH (T292A), and the host U2OS T-REx cells (Mock). The cells were grown in media containing 0.2 mM hydroxyurea for 24 hr. The populations of cells showing the S phase peaks were quantified and indicated below the flow cytometer profiles. See also Figure 1—figure supplement 1. CDK phosphorylates threonine 292, and construction of RAD9-WT or -S291A/T292A(T292A) expressing stable cell lines.

Figure 1.

Figure 1—figure supplement 1. CDK phosphorylates threonine 292, and construction of the RAD9-WT and -S291A/T292A(T292A) expressing stable cell lines.

Figure 1—figure supplement 1.

(A) HEK293A cells were transfected with a plasmid that expresses RAD9-WT-mH (WT) or RAD9-T292A-mH (T292A) under the CMV promoter, cultured in the presence or absence of RO3306 (5 µM, indicated as 3306) or Roscovitin (20 µM, indicated as Ros), and subjected to a fractionation assay. Western blotting analyses using α-myc (RAD9-myc), α-pThr292 (pThr292), α-tubulin (TUBULIN), α-RPA32 (RPA) and α-Histone H1 (Histone H1) are shown. (B), (C) Stable cell lines expressing RAD9-WT-mH (WT) or RAD9-S291A/T292A-mH (T292A) were constructed in HEK293A-T-REx (B) or U2OS T-REx (C) cells. A western blotting analysis was performed to compare the expression levels of endogenous RAD9 and the expressed myc-tagged RAD9. RAD9 was expressed under CMV promoter control via the tetO system. α-RAD9 (Bethyl) was used for the western blot analysis. Doxycyclin (Dox) was added to induce the promoter. Asterisks (*) are nonspecific signals. For the controls, host cell lines (-) were used.