JCS01405 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Orc1p* binds to chromatin, co-immunopurifies with Orc2p and its level is cell cycle-regulated. (A) Western blot analysis of fractionated HeLa cells. S1, cytoplasm; S3, nucleoplasm; P3, chromatin-enriched fraction. Endogenous Orc1p and Orc2p were revealed with specific polyclonal antibodies, Orc1p* was localized with the anti-Flag polyclonal antibody. (B) Western blot analysis of nuclear extracts (NE) prepared from non-transfected HeLa cells (–) or from HeLa cells expressing Orc1-Flag (+). Extracts were probed with the anti-Flag polyclonal antibody and the anti-Orc2 polyclonal antibody. The same extracts were used in co-immunoprecipitation experiment with the monoclonal anti-Flag M2 affinity gel (IP). The presence of Orc1-Flag and of Orc2p in the immunoprecipitate was revealed in western blotting with anti-Flag and anti-Orc2 polyclonal antibodies. (C) HeLa cells expressing Orc1-Flag were arrested in mitosis with nocodazole (Noc) and allowed to recover from the block for 1, 10, 15 and 22 hours. At each time point, aliquots of the whole cell extract were analyzed by western blotting with the anti-Flag antibody. Synchronization was verified following the expression of cyclin A and E. The level of a-tubulin in each sample is also shown.

• Supplemental Figure 2 -

  Fig. S2. Immunolocalization of Orc1p* in NIH-3T3 cells. Exponentially growing NIH-3T3 cells were transfected with pOrc1-Flag/GFP plasmid described in Fig. 4. After 48 hours, cells were fixed and stained with anti-Flag polyclonal and with anti-PCNA monoclonal antibodies. Antibodies were revealed with the TRITC-conjugated anti-rabbit and with the Cy5-conjugated anti-mouse secondary antibodies. Nuclei were stained with DAPI. Transfected cells were revealed by GFP fluorescence. Confocal laser images were taken. (A) GFP-positive, PCNA-positive S-phase nuclei in which Orc1-Flag is not detectable (arrows). (B) GFP-positive, PCNA negative G1 nucleus that expresses Orc1-Flag (arrowhead). 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.

• Supplemental Figure 3 -

  Fig. S3. Orc1p* in nocodazole-arrested cells. NIH-3T3 cells were transfected with pOrc1-Flag/GFP plasmid described in Fig. 4 and synchronized in mitosis with nocodazole as described in Materials and Methods. Shaken-off mitotic cells were spotted onto a poly-lysine coated slide before fixation. Cells were stained with rabbit anti-Flag (c and e) and rat anti-HP1b (b) antibodies. Antibodies were revealed with the TRITC-conjugated anti-rabbit and with the Cy5-conjugated anti-mouse secondary antibodies. Nuclei were stained with DAPI (d). Transfected cells were revealed by GFP fluorescence (a). Confocal laser images were taken. Image e is an over-exposure of the image in c to show the absence of a signal above the background in the GFP-positive cell. This pattern is detectable in more than 95% of GFP-positive cells.

• Supplemental Figure 4 -

  Fig. S4. Immunolocalization of the D151-269 mutant fused to the Flag epitope in HeLa cells. Chromatin was stained with DAPI. Confocal laser images of the same field were taken and merged.

• Supplemental Figure 5 -

  Fig. S5. TSA does not affect binding of Orc1p to chromatin. Western blot analysis of S1 (cytoplasm), S3 (nucleoplasm) and P3 (chromatin) fractions prepared from untreated HeLa cells or from cells grown for 6 hours in the presence of 0.5 mg/ml TSA. Endogenous Orc1p was revealed with a specific polyclonal antibody.