For immunoblots, cells were lysed in UTB (9 M urea, 75 mM Tris-HCl pH 7.5 and 0.15 M β-mercaptoethanol) and sonicated briefly. The Odyssey infrared imaging technology was used (LI-COR Biosciences) and the Odyssey analysis system was used for quantification of the immunoblots. In each case, experiments were carried out in triplicate and a representative blot is shown unless otherwise stated. The antibodies used in this study are listed in the reagent and resource table.
For immunofluorescence, cells were seeded on autoclaved cover glasses (Menzel-Glaser). At the end of each experiment cells were fixed with 4% fixation buffer (4% (w/v) paraformaldehyde in PBS), lysed with 1% PBS-Triton X-100 and then incubated with blocking buffer (2% (w/v) BSA in 0.1% PBS-Triton X-100). Incubation with the appropriate primary and then secondary antibody followed (diluted in 2% (w/v) BSA - 0.1% PBS-Triton X-100) (antibodies used are listed in the reagent and resource table). Cells were visualized using a LSM780 (Carl Zeiss Microscopy Ltd) confocal microscope. At least 100 cells were counted per condition. Due to the presence of 53BP1 foci in the nuclei of unstressed cells, induction of DNA damage was quantified by counting cells with more than six foci. Induction of ssDNA was quantified by counting cells with more than six RPA32 foci.
For immunofluorescence, cells were seeded on autoclaved cover glasses (Menzel-Glaser). At the end of each experiment cells were fixed with 4% fixation buffer (4% (w/v) paraformaldehyde in PBS), lysed with 1% PBS-Triton X-100 and then incubated with blocking buffer (2% (w/v) BSA in 0.1% PBS-Triton X-100). Incubation with the appropriate primary and then secondary antibody followed (diluted in 2% (w/v) BSA - 0.1% PBS-Triton X-100) (antibodies used are listed in the reagent and resource table). Cells were visualized using a LSM780 (Carl Zeiss Microscopy Ltd) confocal microscope. At least 100 cells were counted per condition. Due to the presence of 53BP1 foci in the nuclei of unstressed cells, induction of DNA damage was quantified by counting cells with more than six foci. Induction of ssDNA was quantified by counting cells with more than six RPA32 foci.