BCAP and CD19 regulate Akt activation and B-cell differentiation through their binding to p85α subunit of PI3K. Bone marrow B cells were prepared from CD19^−/−BCAP^−/− mice. The cells were cultured for 2 days with IL-7 and infected with retrovirus vectors carrying the cDNAs of BCAP (BCAP wt), CD19 (CD19 wt), BCAP mutant (BCAP 4YF), or CD19 mutant (2YF). BCAP 4YF and CD19 2YF are mutants in which 4 and 2 YXXM motifs, respectively, were replaced by FXXM. For examining p85α association to CD19, BCAP, and their mutants, Flag-tagged cDNA was introduced via retrovirus vectors into CD19^−/−BCAP^−/− cells. As a negative control, a retrovirus vector without cDNA (mock) was also used. Infected cells were cultured for an additional 2 days with IL-7. The recovered cells were stimulated with anti-Igβ and subjected to Western blotting analysis as described in Figure 2D. The recovered cells were further cultured for 2 days without IL-7 to induce differentiation and were subjected to flow cytometric analysis as described in Figure 2B. The percentage of IgM⁺IgD⁺ cells is indicated in each panel.