Characterization of MIXL protein expression.

(A) Specificities of MIXL antibodies. 293T cells were transfected with wild-type (WT) MIXL and mutant MIXL without the homeobox (ΔMIXL) cloned in the vector pcDNA3.1-MycHis. Forty-eight hours after transfection, cytoplasmic lysates for homeodomain-less mutant MIXL and nuclear lysates for WT MIXL were resolved on a 12% SDS-PAGE gel and transferred to a Hybond P nylon membrane. The blot was probed with the affinity-purified antibody anti-MIXL-N at a dilution of 1:100 and antiserum anti-MIXL-C at a dilution of 1:1000. Both antibodies detected the same full-length MIXL protein at 36 kd and homeodomain-less mutant MIXL at 26 kd. V indicates vector alone; FL, full-length MIXL construct and ΔHD- homeodomain-less construct. (B) MIXL protein is expressed in hematologic malignancies. MIXL proteins were detected in leukemic cells expressing early myeloid markers (ML3, KG1), lymphoid markers (CEM, Peer, CJ) and erythroid markers (K562). The affinity purified antibody anti-MIXL-N was used at 1:100 dilution. The blot was reprobed with the mouse monoclonal antibodies against human histone H1 to quantify loading.