Fig. 2. Focus-formation of RPA in the nuclei incubated with Xenopus egg extract. (A) Demembranated Xenopus sperm nuclei were incubated at 23°C for 30 minutes (a-c), 60 minutes (d-f) or 90 minutes (g-i). RPA resistant to detergent treatment is shown in panels a, c, d, f, g and i. DNA was visualized using propidium iodide (b,e,h). Panels c, f and i are enlarged images of the nuclei indicated by arrowheads in panels a, d and g, respectively. The same nuclei in the `DNA' panels are also indicated by arrowheads. Bars, 50 µm (a,b,d,e,g,h); 20 µm (c,f,i). (B) A detergent-insoluble nuclear fraction was prepared from the nuclei incubated in Xenopus egg extract for 30 minutes, 60 minutes or 90 minutes. The p32 subunit of RPA in the nuclear fraction was visualized by immunoblotting after SDS-polyacrylamide gel electrophoresis. (C) The detergent-insoluble nuclear fraction prepared from 30 minute incubated nuclei was incubated for 10 minutes at 37°C with (lanes 1,2) or without (lanes 3,4) MNase, and the reaction mixture was centrifuged. The resultant supernatant (lanes 1,3) and precipitated (lanes 2,4) fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting.