FIGURE 1.

Acetylated Lys-120-p53 is enriched at chromatin, cytoplasmic, and mitochondrial fractions. A, 18 h following 7.5 J/m² UV exposure, MCF-7 cells were harvested and biochemically fractionated into nuclear fraction (chromatin-bound and -unbound) and cytoplasmic fractions. Subsequently, each fraction was divided into equal aliquots and subjected to immunoprecipitation with antibodies that recognize either any acetylated protein or specifically the p53 acetylated at lysine 120 (AcK120-p53). Precipitates were then analyzed by Western blot with p53-specific antibodies. Input lysates were also blotted with p53, tubulin (cytoplasmic marker), and ORC2 (nuclear marker) antibodies. B, U2OS cells treated with 0.5 μm adriamycin (Adr) for 18 h and untreated U2OS cells were harvested and separated into 2 equal aliquots. One aliquot from each condition was lysed with a CHAPS-based lysis buffer (described under “Experimental Procedures”) to generate whole cell extracts. Cells from the 2nd aliquot were biochemically fractionated to isolate mitochondria (Mito) and subsequently lysed with a CHAPS-based lysis buffer. Following lysis, 80% of mitochondrial and whole cell extracts were subjected to immunoprecipitation with the AcLys-120 antibody. Precipitates were subjected to Western blot with p53 antibodies to quantify p53 acetylated at lysine 120 (top panel). The amount of p53 localized to the mitochondria is relatively small compared with the amount of total p53 in a stressed cell. Therefore, to compare p53 protein levels between the mitochondria and total p53 lysates in the linear range, 1, 5, and 10% of input lysate from each adriamycin-treated mitochondrial extract (bottom 3 panels, lanes 2–4, respectively) and whole cell extracts (bottom 3 panels, lanes 6–8, respectively) were blotted with p53, voltage-dependent anion-selective channel (mitochondrial marker), and ORC2 (nuclear marker) antibodies. Furthermore, 10% of each untreated fraction was also blotted with the antibodies described (lanes 1 and 5). The acetyl-Lys-120 signals (top panel) from each sample were compared with the titrations probed for total p53 (lower panel). The bar graph to the right represents the percentage of p53 in whole cell extracts acetylated at lysine 120 (light gray, 5 ± 1%) versus the percentage of mitochondrial p53 acetylated at lysine 120 (dark gray, 32 ± 8%).