Fig. 5. Anti-Ciz1 antibody detects endogenous Ciz1 in sub-nuclear foci that overlap with sites of DNA replication. (A) Coomassie Blue-stained SDS-polyacrylamide gel showing purified recombinant ECiz1 fragment Nterm442, and western blots of recombinant Nterm442 using anti-Cdc6 antibody V, and anti-Ciz1 antibody 1793. (B) Western blot of 3T3 whole cell extract. Of the two bands detected by anti-Ciz1 antibody 1793 one has the same mobility as p100-Ciz1 recognized by antibody V and the other has an apparent molecular mass of 125 kDa. (C) Immunoprecipitation from 3T3 nuclear extract, using antibody V or anti-Ciz1 1793. Both antibodies precipitate p100, which is recognized by the reciprocal antibody in western blots. p125 is precipitated by antibody 1793, and to a lesser extent by antibody V and these are recognized by 1793 in western blots. Mcm3 is shown as a control. (D) Endogenous Ciz1 (red) in 3T3 cells fixed before (untreated) or after (detergent treated) exposure to TritonX100, detected with anti-Ciz1 antibody 1793. Nuclei are counterstained with Hoechst 33258 (blue). (E) Nuclei isolated for replication experiments contain detergent resistant and detergent soluble Ciz1 protein, detected with anti-Ciz1 1793. (F) Detergent-resistant Ciz1 (red) is present in all nuclei in cycling populations, while detergent resistant PCNA (green) persists only in S phase nuclei. (G) High magnification confocal section showing detergent resistant Ciz1 and PCNA foci, and merged image showing co-localising foci (yellow). White arrows indicate some foci common to both antigens. (H) Line plot of red and green fluorescence across the merged image in G, at the position indicated by an arrow. (I) Cross-correlation plot (Rubbi and Milner, 2000; van Steensel et al., 1996) for green foci compared to red over the whole merged image in G, and (inset) for the marked section after thresh-holding fluorescence at the levels shown in H. The red line in the inset shows loss of correlation when the Ciz1 image is rotated 90° with respect to PCNA. Bar is 10 µM.