FIG. 6.

Cyclin D1-DMP1 complexes do not bind DNA. (A) Purification of Flag-tagged DMP1 and untagged DMP1 complexed to Flag-tagged cyclin D1. Sf9 cells were infected with baculoviruses encoding Flag-D1 (lane 1), Flag-DMP1 (lane 2), or Flag-D1 and untagged DMP1 (lane 6). Flag-tagged proteins in cell lysates were bound to M2 beads and eluted with a competing peptide. Purified Flag-DMP1 was subjected to serial 10-fold dilution (lanes 3 to 5), and equal aliquots of the purified proteins were electrophoretically separated on denaturing gels. Densitometric analysis indicated that the quantity of untagged DMP1 copurified with Flag-D1 was ∼2% of that of Flag-DMP1. As expected, no protein migrating at the position of DMP1 was detected in cells infected with Flag-D1 alone (lane 1). (B) Equal aliquots of the purified protein samples were subjected to EMSA using a ³²P-labeled DMP1-specific oligonucleotide probe. Whereas DNA-probe complexes were detected in 10,000-fold diluted DMP1 samples, no binding was detected when cyclin D1-bound DMP1 was used.