Fig. 4. Orc1p* in S-phase nuclei. HeLa cells were transfected with a plasmid that directs the expression of a dicistronic mRNA in which the sequence of Orc1-Flag is upstream of the GFP. (A) Schematic diagram of the pOrc1-Flag/GFP plasmid. PCMV, cytomegalovirus promoter; IRES, internal ribosomal entry site; SV40 pA, SV40-derived RNA processing site. (B) Cells were co-stained with the anti-Flag polyclonal and anti-PCNA monoclonal antibodies. Antibodies were revealed with the TRITC-conjugated anti-rabbit and Cy5-conjugated anti-mouse secondary antibodies. Nuclei were stained with DAPI. Confocal laser images were taken. The overlay of GFP (visualized in green), Orc1-Flag (red) and PCNA (blue) is shown (merge). The arrows indicate PCNA-positive GFP-positive nuclei in which Orc1-Flag is not detectable. Arrowheads show transfected nuclei expressing Orc1-Flag that are PCNA-negative. To preserve the GFP signal, we skipped Triton extraction before fixation. Under these conditions PCNA staining was detectable not only in S-phase replicative patterns but also in G2-phase cells characterized by intense homogenous staining. Bar, 10 µm. (C) Shows a PCNA-positive, Orc1p*-positive nucleus in early S phase (early-S) and a PCNA-positive, Orc1p*-negative nucleus in mid S phase (mid-S). The merged images show that in early S phase, Orc1p* does not colocalize with PCNA.