FIGURE 3.

PKCβ shRNA selectively diminishes the protective effect of PMA. A, 48 h after transfection with empty vector (ev) or shRNA targeting the indicated PKC isoform, cell lysates from unsorted Jurkat cells were subjected to SDS-PAGE followed by immunoblotting for the indicated PKC isoform. HSP90 served as a loading control. Dashed lines between lanes indicate that intervening lanes from additional time points have been removed to simplify this figure. Numbers below lanes, relative signal intensity of targeted PKC isoform compared with empty vector after correction for Hsp90 loading. B and C, 48 h after transfection of Jurkat cells with 40 μg of empty vector or shRNA targeting the indicated PKC isoform (along with 5 μg of plasmid encoding EGFP), cells purified on Ficoll-Paque step gradients were treated for 5 h with the indicated concentration of PMA along with 30 ng/ml CH-11 (B) or 2.5 ng/ml TRAIL (C). At the completion of the incubation, cells were stained with APC-conjugated annexin V and analyzed by two-color flow cytometry (49) as illustrated in supplemental Fig. S2. The percentage of EGFP⁺ (shRNA-transfected) cells that bound annexin V is indicated. In the absence of CH-11 or TRAIL, fewer than 20% of the cells were apoptotic.