The AML-1/ETO C-terminal corepressor binding domains are required to block PLZF repression.

Transient transfections and reporter assays performed in 293T cells in 12-well dishes. Results are all depicted as fold repression relative to the control plasmid basal expression level. (A) Experiments performed with the IL-3Rα chain reporter. Lane 1, pCDNA 400 ng. Lane 2, PLZF 400 ng. Lane 3, PLZF 400 ng plus AML-1/ETO 300 ng. Lane 4, PLZF 400 ng plus AML-1/ETOΔ^HHR 300 ng. Lane 5, PLZF 400 ng plus AML-1/ETOΔ^HHR/MYND 300 ng. (B-D) Experiments performed with the (GAL4)₅-tk-Luc reporter and GAL4-PLZF in B, GAL4-BTB/POZ in C, and GAL4-RD2 in D. Doses are identical to those in A. Results of all graphs reflect the average of multiple experiments ± SEM. (E,F) 293T cells were transfected with 750 ng each of pCMV-AML-1/ETOΔ^HHR/MYND and pCDNA-PLZF expression vectors. Cells were lysed and subjected to immunoprecipitations and immunoblotting as indicated. (E) Immunoblot with ETO C-terminal rabbit polyclonal antibody. (F) Immunoblot with PLZF mouse monoclonal antibody. Lanes 1 and 2 of both panels correspond to immunoblots of cell lysates and precipitated pellet, respectively. Lanes 3 to 7 correspond to immunoprecipitates as indicated.