Figure 1.

AKT and NF-κB regulate RAG activity in mouse Abl pre-B cells. (A) Representative fluorescence-activated cell sorter plots of the WT and RAG2^−/− mouse Abl pre-B cells transduced with the retroviral RAG-reporter construct consisting of an antisense GFP complementary DNA flanked by a 12-bp spacer RSS and a 23-bp spacer RSS, followed by an IRES-RFP cassette. RAG activity mediates inversional recombination of the antisense GFP gene, which can be quantified by flow cytometry. WT and RAG2^−/− mouse Abl pre-B cells were treated for 96 hours with 10 μM STI571. (B) WT mouse Abl pre-B cells transduced with the RAG-reporter construct stimulated with 5 μM PI3Ki, 2.5 μM AKTi, 2.5 μM IKKβi, or untreated (dimethylsulfoxide [DMSO] vehicle) for 96 hours. GFP histograms (RAG-reporter activity) of transduced cells are shown by gating on RFP⁺ cells. (C) Titration curve for IKKβi and for IKKβi plus 2.5 μM AKTi. RAG-reporter activity (y-axis) of WT mouse Abl pre-B cells is plotted against the concentration of IKKβi (x-axis). RAG-reporter activity of mouse Abl pre-B cells treated with 5 μM STI571 is shown. Cells were treated for 72 hours. A representative example of 3 independent experiments is shown, 4 replicate measurements were performed per experiment, and error bars show means ± standard deviation (SD).