Figure 2.

Cyclin B1 interacts with Ku70 in Ku-heterodimer in vitro and in vivo. (A) Varying quantities of ⁶His-Ku70 monomer (lanes 1–4) and ⁶His-Ku70/SBP-Ku80 dimer (lanes 5–8), purified from insect cell expression system, were incubated with glutathione agarose beads bound to GST or GST-cyclin B1 (expressed in insect cells). The pulled-down complexes were analyzed by anti-Ku70 and anti-SBP tag (for Ku80) immunoblotting (Interaction). The presence of Ku70 and Ku80, as appropriate, in the reaction mixtures was checked by indicated immunoblotting (Input panels). The presence of GST or GST-cyclin B1 was also tested by anti-GST immunoblotting. (B) Immunoprecipitation with antibodies against cyclin B1 and Ku70 were carried out from the extract of HeLa or HEK293 cells arrested at mitosis by nocodazole and the presence of Ku70, Ku80 or Cdc2 in the immunoprecipitates were determined by immunoblotting with appropriate antibodies as indicated.