Fig 2: Immunogold electron microscopy for the detection of 53BP1 (a, b) and WRAP53 (c) in PDDF. 45d post-IR. Immunogold particles of 53BP1 immunoreactivity strongly decorated the network of chromatin fibers throughout the focus. c Similarly, immunogold labeling for WRAP53 was also detected on the fibers. Scale bar: a = 175 nm, b = 150 nm, c = 125 nm

Fig 3: a–c Immunostaining of SGNs nuclei for 53BP1 in combination with DAPI demonstrated the presence of DNA in a PDDF. 45d post-IR. d–l Double immunolabeling for yH2AX in combination with pATM (d–f), and 53BP1 in combination with either Ub-H2A (g–i) or WRAP53 (j–l) demonstrated the colocalization of all these DNA damage signaling and repair factors in PDDF. 45d post-IR. Scale bar: a-l = 5 µm. m–o Western blot analysis of the expression levels of pATM (m), Ub-H2A (n) and WRAP53 (o) in sensory ganglion of non-irradiated and irradiated rats exposed to one, two or thee doses of IR (4Gy each; n = 3 animals per group). Note the induction of pATM after IR, and the dose-dependent substantial increase of both pATM and Ub-H2A protein levels. A moderate increase over the basal levels in control ganglion of WRAP53 was also observed upon IR. The expression of alpha-tubulin band was used as a protein loading control, and the fold increase estimated

Fig 4: WRAP53ß accumulates at DNA double-strand breaks in cells overexpressing this protein. (a) Mock or Flag-WRAP53ß cells were left untreated or irradiated (IR) with 6 Gy, and 1 h later, their soluble proteins were removed by pre-extraction and the cells were then immunostained for WRAP53ß (with WRAP53-1F12 antibody) and ?H2AX. The numbers represent the percentage of 100–200 cells counted whose nuclei contained >10 WRAP53ß or ?H2AX foci. Means±S.D. are shown, n=3. (b and c) After irradiation and pre-extraction as above, Flag-WRAP53ß cells were immunostained for the proteins indicated. The FK2 antibody recognizes conjugated ubiquitin