PML I is a part of the AML1 complex. (A) Purification of the AML1b complex. The complexes were purified from cell lysates prepared from L-G infectants carrying empty vector (mock), stably expressing FLAG-tagged AML1a (AML1a, a splicing variant lacking the C-terminal transcriptional regulatory domain), and FLAG-tagged full-length AML1b proteins (AML1b). The complexes were immunoprecipitated on anti-FLAG antibody-conjugated agarose, and the bound materials were eluted with the epitope peptide. Proteins were resolved by SDS-PAGE and visualized by silver staining. The proteins were identified by mass analysis. (B) The amino acid sequences of peptides derived from the 125-kDa protein specific to the fraction purified from the FLAG-AML1b expressing cells. The protein was identified as PML (accession number NM178087). (C) Detection of PML protein in the AML1 complex. The purified AML1 complexes were analyzed by immunoblot with anti-mouse PML antibody (rabbit polyclonal, Wang et al, 1998). (D) Interaction of endogenous AML1 and PML proteins. Cell lysates were prepared from 5 × 107 K562 cells and then were immunoprecipitated with anti-human PML monoclonal antibody 1B9 (MBL) or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-mouse AML1 rabbit polyclonal antibody.
