HOXA9 and SMAD4 are coexpressed in human AMLs with NUP98-HOXA9 chromosomal translocation or with high levels in expression of HOXA9 as well as in murine primitive hematopoietic cells immortalized by HOXA9 or NUP98-HOXA9. (A) Western blot analysis of BM cells derived from patients with AML (left panel), using BM from AML patients with low-level expression of HOXA9 (HOXA9^low, n = 5), with high-level expression of HOXA9 (HOXA9^high, n = 6), or BM samples from AML patients with chromosomal translocation t(7;11)(p15;p15), resulting in NUP98-HOXA9 cytogenetic translocations (NUP98-HOXA9, n = 4). Normal human BM (n = 2) was used as control. Quantification of gel (right panel) reveals a correlation between SMAD4 and HOXA9 intensity. FAB classification is shown on the top (nd indicates not determined). (B) Representative Western blot showing the coregulation of Smad4 and Hoxa9 proteins in murine Wt HSPCs immortalized by HOXA9 (light blue dots) or NUP98-HOXA9 (pink dots) and presentation of the relative increase compared with control samples (Mig vector, blue dark dots) as seen in the right panel. (C) Representative immunoblots showing stabilization of Smad4 and Hoxa9 proteins in Wt HSPCs immortalized by HOXA9 or NUP98-HOXA9. After immunoprecipitation (IP) with the anti-Smad4 antibody and PAGE in the absence of SDS (native condition), the Smad4-Hoxa9 complexes were analyzed by immunoblot (IB) with anti-Smad4 (left) and anti-Hoxa9 (right) antibodies (1 representative experiment of 2 independent experiments). (D) The immunoprecipitation with anti-Hoxa9 antibody and immunoblot (SDS-PAGE) with the anti-Smad4 antibody confirms the presence of Smad4-Hoxa9 complexes in Wt HSPCs immortalized by HOXA9 or NUP98-HOXA9. (B-D) HSPCs (Lin⁻ cells) were isolated from hematopoietic cells generated at serial passage 2 or 3 using CFU assay (HSPCs immortalized by HOXA9 or NUP98-HOXA9). Control HSPCs (Lin⁻ cells) were isolated from hematopoietic cells generated from first passage (HSPCs not immortalized, transduced with Mig).