Extracting Freely Diffusing Proteins from Cells

To extract proteins that freely diffuse in cells, the cells were permeabilized as described previously (Kimura et al., 2006 (link)). Cells that had been plated on a 35-mm glass-bottom dish 1 day before were chilled on ice, and washed twice with ice-cold PBF [100 mM potassium acetate, 30 mM KCl, 10 mM Na₂HPO₄, 1 mM dithiothreitol, 1 mM MgCl₂, 1 mM adenosine triphosphate (Thermo Fisher Scientific), and 5% Ficoll (Nacalai Tesque)] followed by incubation with ice-cold PBF containing 0.1% Triton X-100 for 5 min. The cells were washed twice with cold PBF and incubated with Cy5-conjugated γ-H2AX Fab and Alexa Fluor 488-conjugated H4K20me2 Fab in PBF for 3–4 h on ice. The laser irradiation assay and observation were performed at 29°C using a confocal microscope (FV-1000), as described above. To compare the levels of endogenous proteins that remained after permeabilization, cells without or with permeabilization were fixed, treated with Triton X-100, and incubated with mouse anti-ATM (0.2 μg/ml; Santa Cruz Biotechnology; G-12) or mouse anti-Ku86 (1 μg/ml; Santa Cruz Biotechnology; B-1) antibody, and then with Cy3-conjugated goat anti-mouse IgG (H+L) (0.5 μg/ml; Jackson ImmunoResearch) and Hoechst 33342, as described above.