HoxA9 is degraded by neutrophil granule proteases. (A) HoxA9 is unstable in myeloblasts but not in epithelial cells. Triton-based lysates of primary murine bone marrow cells transformed by HA-tagged HoxA9 or extracts from 293T cells transfected with the same construct were incubated on ice with samples taken at the indicated time points, inactivated in hot SDS, and analyzed by western blotting. (B) HOXA9 is unstable in human AML cell lines. Extracts from THP1 and Molm13 cells derived from patients with MLL translocations and, therefore, expressing high levels of HOXA9, were extracted as above. Lysates were immediately denatured in SDS or incubated on ice for 10 minutes before western blot analysis for endogenous HOXA9. (C) High concentrations of the weak elastase inhibitor chymostatin inhibit HoxA9 degradation in myeloblast extracts. Triton-based extracts were produced from primary cells transformed by HA-tagged HoxA9 and supplemented with the indicated concentrations of chymostatin before western blotting. Controls were directly boiled in SDS before analysis. (D) Elastase degrades HoxA9. Extracts of 293T cells expressing HA-HoxA9 were supplemented with SDS or with different concentrations of purified elastase or were mixed 1:1 with myeloblast extract, as indicated. Lysates were incubated for 10 minutes on ice before western blotting. (E) HoxA9 is a substrate for elastase, proteinase 3, and cathepsin G. HoxA9-transformed primary HSPCs from Elane^−/− mice were further deleted for Prtn3 and Ctsg by Crispr-based knockout. Lysates of individual cell lines were tested for HoxA9 stability, as above. (F) HoxA9 is stable in EPC triple-knockout myeloblasts. HoxA9-transformed cells from EPC or WT animals were lysed, and the stability of HoxA9 was tested as before.