Figure 4.
Myristylated Akt replaces IL-2 signals transduced via the S region of the IL-2Rβ and induces Bcl-2 and c-myc expression in BAF/3-IL-2Rβ·ΔS cells. (A1) 106 BAF/3-IL-2Rβ or BAF/3-IL-2Rβ·ΔS cells were cultured at the concentration of 0.5 × 106 cells per ml in the absence of IL-2 or IL-3. Twenty-four hours later, the indicated cultures (lanes 2 and 4) were stimulated with IL-2 (100 units/ml) for 10 min. Unstimulated (lanes 1 and 3) and IL-2-stimulated (lanes 2 and 4) cells were lysed in the Nonidet P-40 lysis buffer. One × 106 cells from four independent BAF/3-IL-2Rβ·ΔS lines expressing a hemagglutinin-tagged myristylated Akt (MA1, MA2, MA3, and MA4) were cultured in parallel with the preceding cells also for 24 h in the absence of IL-2 and IL-3, and they were lysed in the same buffer. Western blots of all the lysates were probed with an anti-Bcl-2 rabbit polyclonal antiserum or with the anti-hemagglutinin tag antibody 12CA5. (A2) Five × 106 cells from four independent cultures of myristylated Akt-transfected, MA1, MA2, MA3, and MA4 and two of CMV-6-transfected BAF/3-IL-2Rβ·ΔS cells ΔS1 and ΔS2 were cultured in the presence of IL-2 (100 units per ml). At time 0, all cells were treated with staurosporine (2 μM). Cells were harvested at sequential time points as indicated, and live and dead cells were counted. The percentage of dead cells was calculated as described in Materials and Methods. (B) Western blots of the cell lysates, analyzed in A1, were probed with an anti-c-myc rabbit polyclonal antibody (Upstate Biotechnology) (dilution 1/2,000).