FIGURE 4.

Kpna was associated only with Orc6 that was not associated with other ORC subunits. A, an anti-FLAG immunoprecipitate was prepared from a TK100 extract of 20 × 10⁸ cells that constitutively expressed FH-Orc6. The immunoprecipitate was then fractionated by glycerol gradient centrifugation (4 ml 10–25% glycerol gradient for 5 h at 55,000 rpm and 10 °C, Beckman Ti-60 rotor), and aliquots from each fraction were subjected to Western immunoblotting using the indicated antibody. B, Orc6 is the only ORC subunit bound to karyopherin-α. TK100 extracts were prepared from HeLa-RR cell lines that constitutively expressed one of the six FH-tagged ORC subunits (1, 2, 3, 4, 5, or 6). Anti-FLAG immunoprecipitates were fractionated by SDS-PAGE and immunoblotted using anti-Kpna (upper panel) and anti-FLAG (lower panel) antibodies. Experiments with the six FH-tagged ORC subunits were done at different times, and therefore, separate gels are presented together.