Skip to main content
. 2017 Feb 17;292(16):6461–6467. doi: 10.1074/jbc.M117.777474

Figure 3.

Figure 3.

53BP1-TIRR complex is required for the expression of both 53BP1 and TIRR, and this complex dissociates following DNA damage. A, overexpression of TIRR reduced 53BP1 foci formation following IR. Cells were transfected with constructs encoding tagged TIRR or Nudt15 and treated with 10 Gy of IR. Immunostaining experiments were performed using anti-53BP1 and anti-FLAG antibodies. B, depletion of TIRR decreases 53BP1 protein level. MCF10A cells were infected with control or TIRR-specific gRNAs. Cells were harvested and immunoblotted with the indicated antibodies. C, Nudix motif of TIRR is critical for 53BP1 expression. TIRR knock-out cells were reconstituted with wild-type TIRR or Nudix motif deletion mutant of TIRR. Cell lysates were immunoblotted with indicated antibodies. D, knock-out 53BP1 decreases TIRR protein level. MCF10A cells and MCF10A-derived 53BP1 knock-out cells were treated with or without IR (10 Gy). Cells were harvested and immunoblotted with indicated antibodies. E, IRIF region of 53BP1 is required for TIRR stability. 53BP1 knock-out cells were reconstituted with wild-type 53BP1 or an IRIF deletion mutant of 53BP1. Cell lysates were immunoblotted with indicated antibodies. F–H, 53BP1 and TIRR dissociate after DNA damage. F and G, beads coated with bacterially expressed MBP-TIRR fusion protein were incubated, respectively, with cell lysates containing exogenously expressed SFB-tagged 53BP1 IRIF region mock-treated or treated with ATMi (KU60019) with or without IR (20 Gy) (F) or HeLa cell lysates treated with or without IR (20 Gy) (G). Immunoblotting experiments were carried out using the indicated antibodies. Alternatively, cell lysates with or without IR (20 Gy) were immunoprecipitated with anti-TIRR antibodies and immunoblotted with indicated antibodies (H). Dissociation of 53BP1 with TIRR does not require TIRR modification following DNA damage (I). Beads coated with bacterially expressed MBP-53BP1 IRIF region fusion protein were incubated with cell lysates containing exogenously expressed SFB-tagged TIRR mock-treated or treated with ATMi (KU60019) with or without IR (20 Gy). Immunoblotting experiments were carried out using indicated antibodies. WB, Western blotting.