PU.1 mutant proteins are expressed in leukemic cells.

Whole cell lysates from leukemic cells at diagnosis were analyzed by Western blot for PU.1 expression (upper panel). U937 cells served as a positive control, whereas COS cells were negative for PU.1 expression. AML patients #97 and #103 had an AML-M0 subtype and lacked PU.1 mutations. In contrast, AML-M0 patient #104 carries the heterozygous V105-H264del mutation that encodes a mutant peptide lacking the PEST and Ets domains. This peptide is detected by an amino-terminal PU.1 antibody19 (data not shown), not by the antibody used in this blot, which is raised against a carboxyl terminal epitope deleted in the mutant allele. The peptide detected in patient #104 is that encoded by the wild-type allele, and approximately one-half as much protein is detected as compared to the other samples. Patient #95 is an AML-M4 with no PU.1 mutation. (Lower panel) The same blot was stained for β-tubulin as a loading control. The comparative amount of PU.1 protein was assessed by quantitation on a phosphorimager (Molecular Dynamics) and normalized to β-tubulin.