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. 1999 Oct 26;96(22):12367–12372. doi: 10.1073/pnas.96.22.12367

Figure 5.

Figure 5

Influence of spOrc4pΔN on complex formation. In vitro transcription/translation product of the plasmid encoding the C-terminal region of spOrc4p (Orc4pΔN, lane 1), which was incubated with (lane 2) or without DNase 1 (lane 3), was immunoprecipitated (indicated by IP) with anti-T7 antibody. The spOrc4pΔN was coexpressed in the presence of the five other subunits in an in vitro transcription/translation reaction (lane 4), and the reaction mixture was incubated with (lanes 5 and 6) or without DNase I (1,000 units/ml) (lanes 7 and 8). The products were immunoprecipitated with antibodies against T7 (lanes 5 and 7) or HA (Orc5p, lanes 6 and 8). As controls, transcription/translation reactions that coexpressed five of the subunits but lacked either Orc4 or Orc5 were subjected to immunoprecipitation with anti-T7 antibody (lane 9) or anti-HA antibody (lane 10), respectively. Proteins that bound to the protein A-agarose beads were subjected to SDS/PAGE, after which the gels were dried and autoradiographed.