Fig. 5. Depletion of RP-A, but not of XKu86, inhibits the incorporation of dNTPs in foci of EcoRI-preatreated sperm pronuclei. Double immunofluorescence with affinity-purified rabbit anti-RP-A70 antibodies (RP-A) or anti-XKu86 antibodies (XKu86) and with streptavidin conjugated to FITC to visualize the incorporation of dNTPs (dNTPs). Sperm heads (
4000 heads) pretreated with 0.02 units of EcoRI were incubated with HSS that was immunodepleted with rabbit anti-RP-A antibodies in the presence of biotinylated dATP and dUTP. With RP-A-depleted HSS, no fluorescence signal of dNTPs is produced. (B) As in (A), but the HSS was immunodepleted with anti-XKu86 antibodies. Depletion of XKu86 from the HSS does not inhibit either RP-A or dNTP accumulation in the foci of the digested nuclei. In the merged figure, most fluorescence RP-A and dNTPs foci co-localize. The yellow color is not that apparent in some co-localized foci (arrows) owing to an imbalance between the two color intensities. Cumulative images of 10-12 confocal sections for each fluorescence channel are shown. Bars, 3 µm.
