Fig. 2.

Physical interaction between HoxA11 and Foxo1a originated in the mammalian stem lineage. Note the presence of a band for human, opossum, ancestral eutherian, ancestral therian, and ancestral mammalian HoxA11-V5/His, but there is no band for chicken or ancestral amniote HoxA11-V5/His, indicating that the physical interaction arose in the mammalian stem lineage. The legend in the upper right corner of the figure indicates location/identity of bands shown in panels, including unknown peptides (observed in some panels) that cross-react with the antibody. Asterisks (*) indicate reconstructed ancestral proteins. Flag epitope-tagged Foxo1a and V5/His epitope-tagged HoxA11 mammalian expression vectors were cotransfected into HeLa cells, and nuclear lysates were incubated with anti-Flag agarose (Sigma) overnight. The following day, samples were treated with DNaseI and washed, and protein complexes were eluted with NuPage LDS sample buffer. Protein complexes were resolved by SDS/PAGE and transferred to a PDVF membrane. Blots were probed with anti-Flag (1:100,000; Sigma) and anti-V5 (1:5,000; Invitrogen) antibodies. Experiments were repeated a minimum of three times, and the results presented here are representative of typical findings.