Figure 7. Failure of genetic ablation of p53 in BMKO mice to restore serum Ig levels, Ig class-switching, and B1a cells. (A) Serum IgM (left) and IgG1 (right) levels in unimmunized mice of the indicated genotypes were determined by ELISA. Horizontal bars are the geometric mean. Results shown are from a single experiment involving 6–10 mice per genotype. (B) Purified splenic B cells from mice of the indicated geno- types were stimulated with LPS plus IL-4 for 4 d and the percentage of IgG1+ class-switched B cells was determined by flow cytometry. Numbers indicate the percentage of class-switched B cells among gated B cells. (C) The percentage of B1a cells (CD5+B220+) in the peritoneal cavity of mice of the indicated genotypes was determined by flow cytometry. For B and C, results are representative of two to three independent experiments involving one to three mice per genotype. Mule maintains homeostasis under genotoxic stress conditions through the ATM–p53 axis To examine how Mule regulates p53 protein levels under stress conditions, we treated Mule-deficient MEFs and BMKO B cells with genotoxic agents known to elicit a p53- dependent response. Control MEFs treated with doxorubicin (Dox) exhibited up-regulation of Mule protein as early as 10 min after Dox treatment (Fig. 8 A). p53 protein gradually accumulated in Dox-treated control MEFs and became phosphorylated at Ser18 (Fig. 8 B, left). Consistent with p53 being a Mule substrate (Chen et al., 2005), high levels of p53 protein were present in Mule-deficient MEFs even at steady state (Fig. 8 B, right, top row). However, phosphorylation of this p53 in response to Dox treatment was impaired (Fig. 8 B, right, middle row). Because genotoxic stress induces p53 phosphorylation by activated ATM (Lavin, 2008), we next 180 Figure 8. Abnormal ATM–p53 axis function in Mule-deficient MEFs. (A) Mulefl/y MEFs were treated with 1 µM Dox for the indicated times and extracts were immunoblotted to detect Mule. (B) Mulefl/y and Muledel/y MEFs were left untreated () or treated with 1 µM Dox for the indicated times. Total p53 and phospho-p53-Ser18 (S18-p53) were de- tected by immunoblotting. (C) Mulefl/y and Muledel/y MEFs were treated as in B, and total ATM and phospho-ATM-Ser1987 were detected by immuno- blotting. For phospho-ATM, film exposures of 30 s (row 2) and 5 min (row 3) are shown. Vinculin, loading control. (D) Mulefl/y and Muledel/y MEFs were treated as in C and total Brca1 was detected by immunoblotting. Film exposures of 2 s (row 1) and 30 s (row 2) are shown. (E) Mulefl/y and Muledel/y MEFs were left untreated () or treated for 2 h with 1 µM Dox. Lysates were immunoprecipitated with anti-Mule Ab followed by immunoblotting (WB) with anti–phospho-ATM-Ser1987 Ab. Input, total cell lysate subjected to immunoprecipitation with anti-Mule Ab. For A–E, data are representative of two independent experiments. examined ATM phosphorylation in the absence of Mule. In Dox-treated control MEFs, total ATM protein accumulated with time (Fig. 8 C, top row) and became phosphorylated at Ser1987 by 2 h after stimulation (Fig. 8 C, middle row). However, consistent with their decreased phospho-p53, Dox-treated Mule-deficient MEFs showed much lower levels of phospho-ATM (Fig. 8 C). Another relevant substrate of phospho-ATM is Brca1 (Cortez et al., 1999). In response to Dox treatment, Brca1 accumulated in a time-dependent manner in control MEFs but not in Mule-deficient MEFs (Fig. 8 D). Intriguingly, low levels of phospho-ATM and high levels of Brca1 were present in Mule-deficient MEFs even without genotoxic stress (Fig. 8, C and D), whereas control MEFs showed no detectable phospho-ATM or Brca1 at steady state. To investigate whether there was a physical interac- tion between Mule and ATM that might account for our E3 ligase Mule is essential for B cell homeostasis | Hao et al.