Leukemias comprise the wild-type and mutant Pknox1-transduced cells. (A) Southern blot analysis of proviral integrations in genomic DNA isolated from the bone marrow of leukemic mice. Top panel: DNA was digested with KpnI to release the integrated wild-type or mutant Pknox1 (3.8-4.2 kb) proviruses detectable with a gfp-specific probe. Bottom panel: Clonal analyses of leukemic cell populations. DNA was digested with EcoRI, which cuts inside the provirus and generates a unique DNA fragment for each integration event. Membrane was hybridized with a gfp-specific probe. The identity of the various Pknox1 cDNAs cotransduced together with Hoxa9 is indicated on top, and the numbers represent individual mice analyzed. Note that material obtained from this representative group of mice was used for all analyses shown in this figure. Control lane contains genomic DNA isolated from untransduced bone marrow cells. Digest of the Pknox1-MC retroviral vector is shown as a positive control. (B) Top panel: Northern blot analysis of wild-type and mutant Pknox1 mRNA levels in total RNA isolated from bone marrow cells of leukemic mice. The membranes were hybridized with a 610-bp wild-type Pknox1 cDNA. The double Pknox1 band originates from the presence of an alternative splice site in the retroviral vector.³⁸  Bottom panel: 18S RNA levels are shown as a loading control. Control lane contains RNA isolated from untransduced bone marrow cells. The average AML latency in days is indicated below. (C) Western blot analysis of PKNOX1 levels in nuclear extracts obtained from bone marrow cells of leukemic mice. Top panels: The membranes were hybridized with anti-FLAG to detect expression of the FLAG-tagged PKNOX1 mutants. Bottom panels: Histone H2A levels are shown as a loading control. Lanes 1 to 12 and lanes 13 to 18 contain samples from 2 distinct experiments.