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Figure 4


Fig. 4. Ciz1 resists nuclease extraction. (A) Western blot showing partitioning of Ciz1 in Triton X-100-resistant nuclei after digestion with DNase 1. PCNA is released into the soluble fraction (S). Digestion does not release Ciz1 into the soluble fraction; all Ciz1 isoforms are retained in the pellet (P). (B) Nuclei were visualized by bright-field microscopy at 600x magnification (top panels) or by fluorescence microscopy after staining with Hoechst 33258 (lower panels, blue). Bar, 10 µm. (C) Immunofluorescence image of Ciz1 (red) in DNase 1 or mock-treated nuclei after extraction with 0.5 M or 2 M NaCl as indicated. (D) Ciz1 in nuclei treated with the protein-protein cross-linker DTSP before DNase 1 treatment and extraction with 2 M NaCl. (E) Colocalization of matrix-associated newly synthesized DNA (green) with matrix-associated Ciz1 foci (red) in an early S phase nucleus. Newly synthesized DNA was detected by incorporation of biotinylated-dUTP during in vitro synthesis reactions. Merged image shows very significant overlap (yellow) in this nucleus. Images of Hoechst fluorescence (blue) were taken under equivalent conditions so that they reflect the level of nucleic acid remaining in mock treated and DNase 1 digested nuclei.





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