Figure 5. Reconstitution of Drosophila condensin sub-complexes in vitro.

Drosophila condensin subunits and control proteins were synthesized by coupled in vitro transcription/translation (IVT) either simultaneously in the presence of [³⁵S]methionine (A, C) or in separate reactions containing or lacking [³⁵S]methionine (B, D, E, F). Hot and cold IVT reactions were mixed (B, D, E, F), or used directly (A, C), and were subjected to immunoprecipitations using anti-Flag (A–C) or anti-myc (D–F) antibody-coated beads. The various combinations of synthesized proteins are indicated by plus signs in the top parts of each panel. Proteins in samples of the input extracts (INPUT), supernatants after precipitation (SUP) and eluates from the antibody-coated beads (IP) were separated by SDS-PAGE. The gels were either dried and directly subjected to autoradiography (AR in (A) and (C)), or they were blotted onto nitrocellulose membranes and the proteins were detected by autoradiography (AR) or immunoblotting using anti-Flag (B) or anti-myc (D–F) antibodies.