(B220+IgM+CD21+CD23+) subpopulations by flow cytometry. BrdU incorporation in each subset was detected by flow cytometry using the BrdU-Flow kit according to the manufacturer’s instructions (BD). stimulated with 20 µg/ml LPS plus 100–200 U/ml IL-4 for 3–5 d. The pro- liferation capacity of the stimulated B cells was measured by flow cytometry as denoted by a decrease in CFSE fluorescence intensity. Ca2+ flux. Splenocytes were stained using FITC-conjugated anti-B220 Ab as described in the previous section. 1 × 107 stained cells were incubated at 37°C for 45 min with 5 µg/ml Indo-1 AM (Invitrogen) in RPMI-1640 medium supple- mented with 10% FCS. Indo-loaded cells were resuspended at 5 × 106/ml and kept on ice until use. 500-µl aliquots of Indo-loaded cells were warmed to 37°C for 10 min and a baseline reading was acquired for 1 min. Ca2+ flux induction was triggered by the addition of 20 µg/ml goat anti–mouse IgM (Jackson Immuno- Research Laboratories, Inc.) followed, 7 min later, by addition of 100 nM thapsigargin as a positive control. Data collection was continued for another 5 min. The Ca2+ response was measured based on the ratio of violet (Ca2+ bound) to blue (Ca2+ free) fluorescence as detected by flow cytometry (LSRII; BD). [3H]-thymidine incorporation. Purified splenic B cells were seeded in trip- licate in 96-well U-bottom plates at 105 cells/200 µl in RPMI-1640 contain- ing 10% FCS. Cells were left untreated, or stimulated with various doses of anti-IgM (Jackson ImmunoResearch Laboratories, Inc.), or with anti-IgM plus 1 µg/ml anti-CD40 (BD). At 16, 40, or 64 h after seeding with stimuli, [3H]-thymidine (1 µCi) was added to each well and cells were cultured for an- other 8 h to determine proliferation at 24, 48, or 72 h [3H]-thymidine uptake was assessed using a liquid scintillation -counter (TopCount reader). Humoral immune responses. Mice were immunized i.p. with either 25 µg of the Ti antigen TNP-Ficoll in PBS, or 100 µg of the alum-precipitated Td antigen NP(15)-CG (Biosearch Technologies). Assays were performed using 96-well ELISA plates coated with 20 µg/ml TNP-BSA or 10 µg/ml NP(15)- BSA. Serum samples collected on day 0 (before immunization) and on days 7, 14, and 21 after immunization were serially diluted and applied to the ELISA plates. Bound Abs were detected by the addition of biotinylated goat anti– mouse IgM, IgG1, and IgG3 (Santa Cruz Biotechnology, Inc.). Biotin binding was revealed using streptavidin-HRP and its substrate. Absorbance at 450 nm was measured using a FlexStation Microplate Reader (Molecular Devices). VSV infection. VSV infection was performed as previously described (Junt et al., 2002). In brief, mice were i.v. injected with 105 pfu VSV-Indiana strain (VSV-IND, Mudd-Summers isolate). Virus was propagated on BHK-21 cells at an MOI of 0.01 and plaqued on Vero cells. During survival experiments, the health status of mice was checked twice daily. Upon appearance of clinical signs of VSV replication in the CNS, such as paralysis or scrubby pelt, mice were removed from the experiment and counted as dead. For Ig analyses, serum samples collected on days 0, 2, 4, 6, 10, and 12 after infection were diluted 40-fold in MEM containing 2% FCS. For the de- tection of neutralizing IgM and IgG, various serum dilutions were incubated with 500 pfu VSV-Ind for 90 min followed by transfer to a single cell layer of Vero cells and incubation for 1 h. After addition of an overlay (1:1 MEM/2% methylcellulose), cells were incubated overnight before fixation and staining for 1 h with 0.5% crystal violet (Sigma-Aldrich). The dilution that resulted in a 50% reduction in virus plaques was taken as the neutralizing titer. For IgG titers, serum samples were incubated with an equal volume of 0.1 M -ME in PBS for 1 h at RT before dilution. Before use in experiments, all mouse sera were heated at 56°C for 30 min to inactivate complement. CSR. Purified splenic B cells were cultured for 3–5 d in RPMI-1640 con- taining 10% FCS plus either 20 µg/ml LPS (InVivo Gene) or 20 µg/ml LPS plus 100–200 U/ml IL-4 (PeproTech). The percentages of B cells that had undergone class-switching to production of IgG3 or IgG1 were determined by flow cytometry. CFSE labeling. Purified splenic B cells were incubated with 1 µM CFSE (Invitrogen) in serum-free RPMI-1640 medium for 10 min at 37°C with gentle mixing. After two washes, the labeled cells were seeded at a density of 5 × 106 cells/ml in RPMI-1640 containing 10% FCS. Labeled cells were 184 Generation of Mule-deficient MEFs. MEFs derived from E14.5 embryos arising from the crossing of Mulefl/+ and Mulefl/y mice were transformed with pLPC-E1A-IRES-RasV12 according to a standard protocol. Transformed Mulefl/y MEFs were transduced with His-TAT-NLS-Cre (Peitz et al., 2002), followed by limiting dilution and expansion to screen for single clones that had undergone Cre-mediated deletion of the floxed Mule allele. The elimi- nation of Mule protein in MEFs was confirmed by immunoblotting as described in Immunoblotting and immunoprecipitation. Generation of the Mule-deficient ES cell line. Targeted ES cell clones were transiently transfected with a Cre-encoding plasmid to delete the distal, loxP-flanked exons 76 and 77 of the mouse Mule gene as well as the neomycin resistance gene. DNA samples from neomycin-sensitive clones were digested with StuI and hybridized to probe c, as depicted in Fig. 1. The appearance in these mutants of only a band of 6.9 kb confirmed the total deletion of the tar- geted Mule exons. The elimination of Mule protein in ES cells was confirmed by immunoblotting as described in Immunoblotting and immunoprecipitation. Real-time PCR. Total RNA was extracted from purified splenic B cells using the RNeasy kit (QIAGEN) according to the manufacturer’s protocol. Extracted RNAs were reverse transcribed using random hexamers and the SuperScript III kit (Invitrogen) as per the manufacturer’s protocol. The result- ing cDNAs were diluted 1:10 and served as templates for real-time PCR reac- tions using the Power SYBR-Green PCR Master Mix (Applied Biosystems). The optimization of the real-time PCR reaction was performed according to the manufacturer’s instructions but was scaled down to a 10-µl final reaction volume containing 500 nM forward primer and 500 nM reverse primer. The PCR reaction mixtures were preincubated at 50°C for 2 min, followed by de- naturation at 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min, using the ABI 7700HT Fast Real-Time PCR System (Applied Biosys- tems). Data were analyzed using SDS software (Applied Biosystems). Primer sequences were as follows: p21 forward, 5-GGCCCGGAAC- ATCTCAGG-3; p21 reverse, 5-AAATCTGTCAGGCTGGTCTGC-3; Puma forward, 5-TCACCCTGGAGGGTCATGTA-3; Puma reverse, 5-GCGGGTGTAGGCACCTAGT-3; Noxa forward, 5-ATCAAGGGC- TGAAGGGATTT-3; Noxa reverse, 5-AGAGAGGCACCTGGGA- TATG-3; Mdm2 forward, 5-CCAGCTTCGGAACAAGAGAC-3; Mdm2 reverse, 5-GTCGTTTTGCGCTCCAAC-3; Bax forward, 5-TG- GAGATGAACTGGACAGCA-3; Bax reverse, 5-GAAGTTGCCATCA- GCAAACA-3; p53 forward, 5-ACTGCATGGACGATCTGTTG-3; and p53 reverse, 5-GTGACAGGGTCCTGTGCTG-3. Each sample was assessed in triplicate. Relative mRNA levels were normalized to the house- keeping gene 18S or the actin mRNA and calculated using the comparative threshold cycle method (2Ct). Genotoxic stimuli. For culture under stress conditions, purified splenic B cells were incubated at 37°C in RPMI-1640 containing 10% FCS in the presence or absence of etoposide (Sigma-Aldrich) or dexamethasone (Sigma- Aldrich) at the concentrations indicated in the figures. Alternatively, cells were subjected to 5 Gy -irradiation for the times indicated in the figures. MEFs were cultured at 37°C in DME containing 10% FCS in the presence or absence of 1 µM Dox (Sigma-Aldrich) for the times indicated in the figures. Proteins were analyzed by immunoblotting as described in Immunoblotting and immunoprecipitation. For apoptosis assays, purified splenic B cells were cultured for 20–24 h with the concentrations of etoposide indicated in the Figures. Cells were harvested and stained with Annexin V plus propidium iodide and analyzed by flow cytometry using a standard protocol. Statistical analyses. The Student’s t test with tail 1 and type 3 was used for statistical analyses. Values are expressed as the mean ± SD. For statistical significance: *, P < 0.05; **, P < 0.005; ***, P < 0.0005. E3 ligase Mule is essential for B cell homeostasis | Hao et al.