53bp1 antibody

LAPC4-KD cells (1 × 10⁶ cells/well) were plated on glass coverslips in 6-well plates. After treatment, cells were washed and fixed for immunofluorescence staining as described previously [32 (link)]. Briefly, the prepared samples were probed with phospho-histone-γ-H2AX antibody (Ser139) (Millipore) and p53-binding protein 1 (53BP1) antibody (Santa Cruz) for 2h followed by incubated with Alexa Fluor 488-conjugated anti-mouse antibody and Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen) for 1 h. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (0.2 μg/ml) for 10 min. The stained cells were analyzed under a fluorescence microscope (Carl Zeiss, Göttingen, Germany) with a 63× objective (oil immersion, aperture 1.3).

Untransfected wild-type and PNKP^−/− HeLa cells, as well as stably transfected HeLa PNKP^−/− cells were exposed to 5 Gy γ radiation. 1, 6, 24 h after irradiation, cells were fixed and underwent immunofluorescence staining with 53BP1 antibody (Santa Cruz #sc-517281). Z-stack images were acquired using a Zeiss confocal LSM 710 microscope. For each time point, 120–200 cells from each group were analyzed using Imaris 9.5 software (Bitplane, Belfast, GB).