p300 increases the DNA binding ability of Fli1 through HDAC1-dependent deacetylation of lysine 380. A.

<p>293T cells were transfected with pCTAP wild type Fli1 along with the indicated HAT proteins, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). In order to visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of HAT proteins in cell lysates were determined by Western blotting. <b>B.</b> Whole cell lysates prepared as described above were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-coupled agarose beads. The levels of Flag-tagged proteins and Fli1 in cell lysates were determined by Western blotting. <b>C.</b> 293T cells were transfected with pSG5 wild type Fli1 or Fli1 K380Q mutant along with the p300 expression vector, and incubated for 48 hours. <b>D.</b> 293T cells were transfected with pCTAP wild type Fli1 along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using SA beads, followed by immunoblotting using anti-HDAC1 antibody. To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of ectopically expressed p300 and endogenous HDAC1 in cell lysates were determined by Western blotting with anti-Flag antibody and anti-HDAC1 antibody, respectively.