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Figure 3


Fig. 3. Endogenous Ciz1 resists salt extraction. (A) Western blot of total protein (T) from cycling NIH3T3 cells after separation using 8% SDS-PAGE. The Triton X-100 soluble fraction (S) and insoluble fraction (P) are also shown. Endogenous Ciz1 isoforms (p100, p125a and p125b) are detected with Ciz1 polyclonal antibody 1793. Mcm2 is shown as a control. (B) Sequential extraction of Triton X-100-resistant nuclei with increasing NaCl in CSK buffer. The protein present in the supernatant fraction (SN) is shown. PCNA was used as the control. (C) Ciz1 in extracted nuclei detected by immunofluorescence (red). Nuclei are counterstained with Hoechst 33258 (blue). (D) Equivalent numbers of Triton X-100-resistant nuclei from a cycling population, a population treated with thymidine for 24 hours prior to harvesting, and a quiescent population of NIH3T3 cells were sequentially extracted with 0.5 M and 2 M NaCl. Intervening wash steps, with 0.5 M NaCl are marked with W. A significant proportion of Ciz1 p100 isoform from all three populations is resistant to 2 M NaCl, remaining in the pellet fraction (P) after extraction. SN, supernatant fractions.





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