Endogenous p73α is recruited onto apoptotic target genes in vivo in response to ATO. (A) A chromatin immunoprecipitation assay was performed from either untreated or NB4 and K562 leukemic cells treated with PD98059, ATO, and PD98059 plus ATO. gDNA obtained from untreated or treated cells was used to normalize the DNA to immunoprecipitation. An anti-p73 (H79 polyclonal antiserum from Santa Cruz Biotechnology) or control unrelated antibodies were used. Immunoprecipitated material was amplified using primers specific for p21 or p53AIP1 or bax promoters. (B) NB4 or K562 cells, after 3 hours of pretreatment with PD98059, were incubated with the indicated concentrations of ATO. Expression of PARP, p53AIP1, and Bax was revealed after 24 and 48 hours in NB4 and after 24, 48, and 72 hours of treatment in K562 cells. (C) Levels of p21 protein were assessed by immunoblotting after 24 hours in NB4 and after 48 hours of treatment in K562 cells. (D) Expression of Bcr-Abl after 24 hours of treatment. Cell lysates were analyzed by immunoblotting analysis using a mouse monoclonal anti-PARP (F2; Santa Cruz Biotechnology), rabbit polyclonal anti-p53AIP1 (CT; AnaSpec, San Jose, CA), rabbit polyclonal, anti-Bax (Cell Signaling Technology), horseradish peroxidase conjugate anti-p21WAF1/CIP1 (Santa Cruz Biotechnology), mouse monoclonal anti-c-Abl (24-11; Santa Cruz Biotechnology), and goat polyclonal antihuman actin (Santa Cruz Biotechnology). Antiactin immunoblotting (Santa Cruz Biotechnology) was performed as loading control. AS = ATO.
