FIG. 4.

HOXD13 interacts with the pre-RC protein CDC6. (A) Coimmunoprecipitation using in vitro-transcribed/translated ³⁵S-labeled HA-HOXD13 and CDC6. In the left panel, HA-HOXD13 alone or HA-HOXD13 plus CDC6 was immunoprecipitated with the anti-CDC6 antibody. In the right panel, CDC6 alone or CDC6 plus HA-HOXD13 was immunoprecipitated with the anti-HA antibody. (B) For the upper panel, coimmunoprecipitations were performed using Cos7 whole-cell extracts expressing HA-HOXD13 and using the anti-CDC6 (α-CDC6) or control immunoglobulin G (IgG) antibodies. The immunoprecipitated CDC6 and HA-HOXD13 proteins were revealed by Western blotting (WB) using anti-CDC6 (α-CDC6) and anti-HA (α-HA) antibodies, respectively. An asterisk indicates a nonspecific band revealed by the anti-CDC6 antibody. For the middle panel, coimmunoprecipitations were performed using LD13IΔN-transduced HEK nuclear extracts and anti-HOXD13 (α-D13) or IgG antibodies. For the lower panel, coimmunoprecipitations were performed using HEK293 nuclear extracts and anti-HOXD13 or IgG antibodies. (C) Coimmunoprecipitation using in vitro-transcribed/translated ³⁵S-labeled HA-HOXD13 and Orc2. For the upper panel, Orc2 alone or Orc2 plus HA-HOXD13 was immunoprecipitated with the anti-HA antibody. For the lower panel, coimmunoprecipitations were performed using Cos7 whole-cell extracts expressing Myc-Orc2 alone, or Myc-Orc2, and HA-HOXD13 and using the anti-HA (α-HA) antibody. HOXD13 and Orc2 were revealed by immunoblotting using anti-HA and anti-Myc antibodies, respectively. (D) GST-pulldown assay using bacterially produced GST and a GST fusion with the HOXD13 homeodomain/C-terminus region (GST-D13HD) (lower panel). Resins were challenged with in vitro-transcribed/translated ³⁵S-labeled CDC6 (upper panel) or Orc2 (central panel). Retained material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed by autoradiography. (E) For the upper panel, coimmunoprecipitations were performed using nuclear extracts from HEK293 cells transfected with increasing amounts of the gemininΔDB expression vector (pSport-GMNΔDB). Immunoprecipitations were performed using anti-HOXD13 (α-D13) or control IgG antibodies. For the lower panel, control Western blots (WB) using nuclear extracts from transfected HEK293. L, load; B, bound; Ig, immunoglobulin heavy chains. An asterisk indicates nonspecific bands.