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Fig. 2. Distribution of Orc1p* in different mammalian cell lines. (A) Exponentially growing human HeLa cells were transfected with either Orc1-Flag or Orc1-GFP. After 48 hours, cells were fixed and processed for conventional microscopy. The distribution of Orc1-Flag was revealed with the rabbit anti-Flag antibody and a TRITC-conjugated anti-rabbit secondary antibody. The localization of Orc1-GFP was revealed by GFP fluorescence. Cells were counterstained with DAPI. The distribution of Orc1-GFP was also determined in transfected monkey COS7 and mouse NIH-3T3 cells. (B) Asynchronous NIH-3T3 cells were transfected with Orc1-Flag and after 48 hours were fixed and stained with the rabbit anti-Flag and the rat anti-HP1ß antibodies. Antigen-antibody complexes were revealed with a TRITC-conjugated anti-rabbit secondary antibody and with a Cy5-conjugated anti-rat secondary antibody (visualized in green). Nuclei were stained with DAPI. Confocal images of the same field were taken. Bar, 10 µm.
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