Skip to main content
. 2021 Jan 6;49(2):928–953. doi: 10.1093/nar/gkaa1232

Figure 5.

Figure 5.

SUMOylation of CtIP in S phase is dependent on the E3 SUMO ligase PIAS4. (A) HeLa His10-SUMO-2 cells were transfected twice 24 h apart with siRNAs targeting either PIAS1 (at 50 nM), PIAS4 (at 40 nM), or CBX4 (at 40 nM), or mock transfected, while being synchronized by double thymidine block. 48 h after the first transfection, the cells were released for 3 h to approach mid-S phase, portioned into input control and His PD fractions, and processed for whole cell lysis or Ni-NTA affinity purification (top panel). A second run (bottom panel) was performed where the input and His PD portions were normalized to accommodate for alterations in CtIP expression resulting from the depletion of the various E3 SUMO ligases. Shown is a representative result of three independent experiments. (B) As in (A), but using the portion of cells processed for DNA content analysis. Shown are the means of three independent experiments. (C) HeLa His10-SUMO-2 cells were left asynchronous or synchronized to S phase. 24 h prior to harvest, they were transfected with FLAG-tagged human PIAS4 or mock transfected. The cells were portioned and processed as input controls or for His PD. Shown is a representative result of two independent experiments. (D) U-2 OS cells were transfected with GFP-CtIP or GFP empty vector and processed for IP of GFP. Prior to IP, a portion of lysate was saved as an input control. Shown is a representative result of at least four independent experiments. (E) U-2 OS cells stably expressing GFP-CtIP were left asynchronous or synchronized to S phase and processed for IP of GFP. U-2 OS cells transfected with GFP empty vector served as a control. Shown is a representative result of at least four independent experiments. (F) Asynchronous U-2 OS cells stably expressing GFP-CtIP were treated for 4 h with HU at the indicated concentrations or 0.8% H2O (vehicle control) and processed for IP of GFP. U-2 OS cells transfected with GFP empty vector served as a control. Shown is a representative result of at least four independent experiments. * indicates a non-specific immunoreactive band. Geminin and Cyclin D1 are markers for S and G1 phases, respectively.