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. 2014 Oct 27;33(23):2860–2879. doi: 10.15252/embj.201489017

Figure 6. CtIP is degraded in response to DNA damage in G2 phase in an APC/C-dependent manner.

Figure 6

  1. U2OS cells were synchronized using a single thymidine block with 2 mM thymidine for 18 h. Seven hours after release, cells enriched in G2 phase were either fixed directly (lane 1) or exposed to low (2 Gy) or high dose (10 Gy) of IR and harvested at the indicated time points following irradiation. Where indicated, cells were treated with the APC inhibitor proTAME (20 μM) immediately after irradiation. Cells were stained with DAPI and analyzed by flow cytometry.
  2. Cells were treated as in (A) and lysed in RIPA buffer for immunoblotting with the indicated antibodies. The anti-RPA2 blot was reprobed with anti-pRPA2-S4/S8 antibody. The arrow indicates leftover signals of the unmodified RPA2 protein. The asterisk indicates hyperphosphorylated RPA2.
  3. HEK293 cells were transfected with HA-tagged ubiquitin (“HA-Ub”). Thirty hours post-transfection, cells were synchronized using a single thymidine block with 2 mM thymidine for 18 h. Five hours after the release, cells enriched in late S/G2 phase were either lysed directly (lane 1), or further incubated for 5 h in the presence of CDK1 inhibitor RO-3306 (9 μM) to keep cells in G2 (lane 2), or irradiated with 10 Gy in the absence (lane 3) or presence (lane 4) of the APC inhibitor proTAME (20 μM) and lysed after 5 h. Where indicated, cells were treated with MG-132 (20 μM) for 5 h before lysis. Samples were then further processed for immunoprecipitation using a polyclonal anti-CtIP antibody as indicated in Materials and Methods. Cell cycle profiles of corresponding samples are indicated on the right.
  4. U2OS Flp-In T-REx cells were induced to express GFP-CtIP-wt and transfected with CtIP siRNA. Cells were subsequently synchronized by thymidine incubation for 24 h and released from thymidine for 8 h. At 8 h after release, cells were treated with DMSO or proTAME (12 μM), subsequently irradiated with 10 Gy, and imaged using fluorescence time-lapse microscopy. Representative stills of GFP and DIC movies are presented in Supplementary Fig S6C. Averages and standard deviations of total nuclear GFP intensity are indicated from 9 and 12 movies for DMSO-treated and proTAME-treated cells, respectively.