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. 2008 Apr 21;181(2):269–280. doi: 10.1083/jcb.200708155

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Copyright © 2008, The Rockefeller University Press

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Figure 5. — LB3 is present on demembranated sperm chromatin and is closely associated with PCNA during nuclear assembly. Demembranated sperm chromatin was exposed to hS, prepared for immunofluorescence with antibodies directed against LB3 and PCNA, and stained with Hoechst 33342 (A–E). Note that PCNA is absent, whereas LB3 is present and frequently appears to be helically wound around the chromatin (D and E). After the addition of X. laevis interphase extracts to sperm head chromatin for 5 min, LB3 is reorganized into patches and foci associated with chromatin (G, I, and J). In addition, PCNA is now present (H) and closely associated with LB3 (I and J). Enlargements of the regions in the boxes in the merged images (D, DNA and LB3; and I, LB3 and PCNA) are shown (E and J, 5.5× view). Bars, 5 μm. (K) The presence of LB3 and the absence of PCNA on demembranated sperm chromatin is also seen by immunoblotting. Demembranated sperm heads in the amount of 50,000 (lane 1), 100,000 (lane 2), or 200,000 (lane 3) were separated by SDS-PAGE, transferred to nitrocellulose, and probed for the presence of LB3 and PCNA. LB3 is seen in all lanes and is quantified relative to lane 3, but no PCNA could be detected.