Fig. 3. RPA34 foci do not form in the presence of p21 but assemble in the absence of nuclear membrane formation. (A) Sperm nuclei were incubated in a calcium-activated mock () or p21-treated () extract. DNA replication was monitored by incorporation of [³²P]dCTP (Materials and Methods). (B) The same experiment as in A except that biotin-dUTP was used to follow DNA synthesis. Samples were treated with 0.3% Triton X-100 before formaldehyde fixation, and immunofluorescence analysis was carried out with the RPA34 monoclonal antibody at the initiation (30 min) or elongation stage (60 min) of DNA replication. (C) Sperm nuclei were incubated in low-speed egg extracts (LSE) or high-speed egg extracts (HSE) as above. DNA was stained with Hoechst, RPA34 was detected using the monoclonal antibody and -H₂AX, a marker of DNA damage, with a specific polyclonal antibody (see Materials and Methods; Fig. S2, see supplementary material).