Figure 6
Figure 6. Panobinostat induces degradation of A/E9a and A/E. (A) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with DMSO vehicle (D) or 16 nM panobinostat (P) for the indicated time using antibodies to AML1, GFP, and acetylated histone H3. β-actin served as loading control. The results shown are representative of at least 3 independent experiments. (B) Western blot analysis of whole-cell lysates prepared from Kasumi cells treated in vitro with DMSO vehicle (D) or 8 nM panobinostat (P) for the indicated time using antibodies to AML1 and acetylated histone H3. Total histone H3 and β-actin served as loading controls. The results shown are representative of at least 3 independent experiments. (C) Western blot analysis of whole-cell lysates prepared from primary t(8;21) AML cells treated in vitro with DMSO (D) or indicated concentrations (nM) of panobinostat (P) for 6 hours using an antibody to AML1 (upper panel). Membrane was stripped and reprobed for p42 as loading control (bottom panel). (D) Western blot analysis of whole-cell lysates prepared from a different primary t(8;21) AML sample as that shown in panel C treated in vitro with DMSO (D) or 8 nM panobinostat (P) for the indicated time using an antibody to AML1 (upper panel). Membrane was stripped and reprobed for anti–acetyl-tubulin antibody (bottom panel). (E) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with DMSO vehicle (D) or 16 nM panobinostat (P) for 24 hours with addition of 5 µM MG132 for the final 4 hours (lanes 1 and 2) using antibodies to AML1, acetylated histone H3, ubiquitin, and GFP. The results shown are representative of at least 3 independent experiments. (F) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with vehicle (D), 16 nM panobinostat (P), or 100 nM of the Hsp90 inhibitor 17-AAG for 24 hours using antibodies to AML1. β-actin served as loading control. The results shown are representative of at least 3 independent experiments. (G) Western blot analysis of the interaction between A/E9a and Hsp90 in A/E9a;NrasG12D leukemic cells treated with vehicle (D) or 16 nM panobinostat (P) for 4 hours. A control mouse immunoglobulin G (IgG) and antibody to Hsp90 were used for immunoprecipitation; antibodies to AML1 and Hsp90 were used for western blotting. The results shown are representative of at least 3 independent experiments.

Panobinostat induces degradation of A/E9a and A/E. (A) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with DMSO vehicle (D) or 16 nM panobinostat (P) for the indicated time using antibodies to AML1, GFP, and acetylated histone H3. β-actin served as loading control. The results shown are representative of at least 3 independent experiments. (B) Western blot analysis of whole-cell lysates prepared from Kasumi cells treated in vitro with DMSO vehicle (D) or 8 nM panobinostat (P) for the indicated time using antibodies to AML1 and acetylated histone H3. Total histone H3 and β-actin served as loading controls. The results shown are representative of at least 3 independent experiments. (C) Western blot analysis of whole-cell lysates prepared from primary t(8;21) AML cells treated in vitro with DMSO (D) or indicated concentrations (nM) of panobinostat (P) for 6 hours using an antibody to AML1 (upper panel). Membrane was stripped and reprobed for p42 as loading control (bottom panel). (D) Western blot analysis of whole-cell lysates prepared from a different primary t(8;21) AML sample as that shown in panel C treated in vitro with DMSO (D) or 8 nM panobinostat (P) for the indicated time using an antibody to AML1 (upper panel). Membrane was stripped and reprobed for anti–acetyl-tubulin antibody (bottom panel). (E) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with DMSO vehicle (D) or 16 nM panobinostat (P) for 24 hours with addition of 5 µM MG132 for the final 4 hours (lanes 1 and 2) using antibodies to AML1, acetylated histone H3, ubiquitin, and GFP. The results shown are representative of at least 3 independent experiments. (F) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with vehicle (D), 16 nM panobinostat (P), or 100 nM of the Hsp90 inhibitor 17-AAG for 24 hours using antibodies to AML1. β-actin served as loading control. The results shown are representative of at least 3 independent experiments. (G) Western blot analysis of the interaction between A/E9a and Hsp90 in A/E9a;NrasG12D leukemic cells treated with vehicle (D) or 16 nM panobinostat (P) for 4 hours. A control mouse immunoglobulin G (IgG) and antibody to Hsp90 were used for immunoprecipitation; antibodies to AML1 and Hsp90 were used for western blotting. The results shown are representative of at least 3 independent experiments.

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