Figure 1. Generation of floxed Mule mice. (A) Targeting scheme. (1) Restriction map of the WT genomic mouse Mule locus. Probes used to verify targeting events are indicated as a, b, and c. The expected sizes of the restriction fragments are shown. B, BamHI; S, StuI. (2) Targeting vector (TV) con- struction. The loxP-flanked neomycin resistance gene (neo) was used as a selection marker during ES cell culture. Exons 76 and 77, which encode the HECT domain essential for Mule’s E3 ubiquitin ligase activity, were flanked by loxP sites (triangles). (3–5) The structure of the targeted Mule locus (3), the targeted locus after removal of neo (4), and the locus after deletion of the floxed exons (5) are shown, including the sizes of diagnostic restriction frag- ments. (B) Southern blot analysis of ES cell clones. Genomic DNA from WT E14K cells and two mutant clones (14D1 and 19C5) were digested with BamHI and hybridized with probe (a) to verify the targeting event. (C) Southern blot analysis of genomic DNA from agouti mice carrying the WT or targeting allele (Mule3P/+) as indicated in A3. DNA was digested with StuI and hybridized with probe c to verify germline transmission. (D) Southern blot analysis of genomic DNA from mice carrying the deleted Mule allele (Muledel/+) as indicated in A5. DNA was digested with StuI and hybridized with probe c. cells and thus disrupt homeostasis, p53 protein stability is tightly regulated by posttranslational modifications such as phosphorylation, acetylation, sumoylation, neddylation, meth- ylation, glycosylation, and ubiquitination (Brooks and Gu, 2003; Meek and Anderson, 2009). In resting cells, the ubiqui- tination of p53 by E3 ubiquitin ligases maintains p53 protein at low levels. In contrast, when a cell is exposed to genotoxic stress, p53 is phosphorylated at serine 15 (Ser18 in mouse) by activated ataxia telangiectasia mutated (ATM), a Ser/Thr protein kinase belonging to the PIKK (PI3K [phosphoinositide 3-kinase]-related protein kinase) family (Banin et al., 1998; Canman et al., 1998; Khanna et al., 1998). If the damage to 174 the cell’s DNA cannot be repaired, ATM-mediated phos- phorylation stabilizes the p53 protein such that it induces the apoptosis of the damaged cell. A HECT-containing E3 ubiquitin ligase called Mcl-1 ubiquitin ligase E3 (Mule; also known as Huwe1, ARF-BP-1, and Lasu1) has been reported to mediate the polyubiquitina- tion and degradation of p53 (Chen et al., 2005), among other substrates (Adhikary et al., 2005; Liu et al., 2005b; Zhong et al., 2005; Hall et al., 2007; Herold et al., 2008; Zhao et al., 2008; Parsons et al., 2009; Yin et al., 2010). The X-linked Mule gene encodes a ubiquitously expressed 4,374-aa protein that contains multiple domains, including BH3. Mule is highly E3 ligase Mule is essential for B cell homeostasis | Hao et al.