observations, protein lysates from control MEFs that were left untreated or treated with Dox for 2 h were subjected to immunoprecipitation with anti-Mule Ab followed by anti– phospho-ATM (S1987) immunoblotting. The results showed that Mule was able to interact with phospho-ATM (Ser1987) after Dox treatment (Fig. 8 E, left). The specificity of this interaction was confirmed in a parallel immunoprecipitation experiment using Mule-deficient MEFs (Fig. 8 E, right). As we are aware that the interpretation of p53-related ex- periments in E1A transformed MEFs may be equivocal, we extended our investigations in primary B cells. We exposed control and BMKO splenic B cells to two different genotoxic agents. Consistent with our MEF analyses, Mule was up- regulated in control splenic B cells as soon as 10 min after ex- posure to -irradiation (unpublished data). Although our earlier data showed that p53 protein was undetectable in con- trol splenic B cells at steady state (Fig. 6 A), p53 protein was JEM Vol. 209, No. 1 A r t i c l e Figure 9. Abnormal ATM–p53 axis function in Mule-deficient B cells. (A) Purified splenic control and BMKO B cells were treated with -irradiation (5Gy) for 2 h (left), or 10 µM etoposide for 3 or 6 h (right), followed by immunoblotting to detect phos- pho-p53-Ser18 (S18-p53). (B) Purified splenic litter- mate control and BMKO B cells were -irradiated (5 Gy) followed by immunoblotting to detect phos- pho-ATM-Ser1987 (S1987-ATM) at the indicated times after irradiation. (C) Purified splenic control and BMKO B cells were treated as in B. Total Brca1 was detected by immunoblotting. (D) Purified splenic control and BMKO B cells were treated with 10 µM etoposide (Etop) for 6 h and degradation of Mcl-1 was monitored by immunoblotting. M/A is the ratio of Mcl-1 protein to actin protein as determined by densitometry (the ratio in unstimulated controls was defined as 1). (E) Purified splenic littermate control and BMKO B cells were treated with 10 µM etoposide for 2, 4, or 6 h followed by detection of cleaved (activated) caspase-3 by flow cytometry. Numbers are the percentage of B cells positive for cleaved caspase-3. Results are representative of a single experiment involving three mice per geno- type. (F) Purified splenic littermate control and BMKO B cells were treated with the indicated doses of etoposide or dexamethasone (Dex) for 20–24 h and viability was determined by flow cytometric analysis of Annexin V versus propidium iodide or 7AAD (7-amino-actinomycin D) expression. Left, representative flow cytometry plot. Numbers are percentages of viable cells. Right, quantitation of viability. Results are the combined mean ± SD of three to five independent experiments each involv- ing one to three mice per genotype (**, P < 0.005; ns, not significant). For A–D, results are representa- tive of two to three independent experiments. both stabilized and efficiently phosphory- lated at Ser18 in control B cells subjected to -irradiation for 2 h (Fig. 9 A, left). How- ever, neither stabilization nor Ser18 phos- phorylation of p53 (phospho-p53-Ser18) was observed in -irradiated BMKO B cells (Fig. 9 A, left). Similarly, control B cells (but not BMKO B cells) treated with etoposide accu- mulated phospho–p53-Ser18 (Fig. 9 A, right). In -irradiated control B cells, total ATM protein accumulated with time and became phosphorylated at Ser1987 by 60 min after stimula- tion (Fig. 9 B, left). In contrast, consistent with their decreased phospho-p53-Ser18, -irradiated BMKO B cells showed much lower levels of both total ATM and phospho-ATM (Fig. 9 B, right). As we had observed for MEFs (Fig. 8 C), spontaneous phosphorylation of ATM at Ser1987 occurred in BMKO B cells (but not in control B cells) even in the absence of genotoxic stress (Fig. 9 B, middle row). Again replicating our observations in stressed MEFs, Brca1 protein readily ac- cumulated in -irradiated control B cells but not in BMKO B cells (Fig. 9 C). Thus, Mule is required for efficient phosphory- lation of ATM and induction of some of its substrates. 181