Apoptotic doses of ATO induce TAp73α acetylation and MEK1 inhibition promotes TAp73α tyrosine phosphorylation in leukemic cells. (A) Leukemic cells were pretreated with either DMSO or PD98059 (40 μM) for 3 hours and then treated with ATO (1 μM, NB4; or 2 μM, K562) for 24 hours. Lysates from treated cells were subsequently immunoprecipitated with a rabbit polyclonal anti-p73 (H79; Santa Cruz Biotechnology) or with a control antibody from lysates of NB4 and K562 treated with ATO. The immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using a rabbit polyclonal antiacetylated lysine (Upstate Biotechnology, Lake Placid, NY). To evaluate the relative levels of p73 acetylation, bands were subjected to densitometric scanning using TINA 2 software (Raytest Isotopenmessgerate, Staubenhardt, Germany). (B) Leukemic cells were pretreated with either DMSO or PD184352 (1 μM) for 3 hours and then treated with ATO for 2 hours (1 μM; NB4) or for 24 hours (2 μM; K562). Extracts from mock and treated NB4 and K562 cells were immunoprecipitated with a rabbit polyclonal anti-p300 (N-15; Santa Cruz Biotechnology) or a rabbit polyclonal anti-p73 (H79; Santa Cruz Biotechnology) or with a control antibody. The immunoprecipitates were analyzed by SDS-PAGE and immunoblotted using rabbit polyclonal anti-p300 antibodies (N-15; Santa Cruz Biotechnology; subpanel i), or a mouse monoclonal antiphosphotyrosine antibody (clone 4G10; Upstate Biotechnology; subpanel ii). A 1:5 aliquot of the immunoprecipitated material was immunoblotted with the anti-p73 monoclonal antibody (clone 1288; Imgenex, San Diego, CA; subpanel iii).