FasL is implicated in maintaining high levels of Bim expression in normal human T-cell blasts. (A) Day-6 T-cell blasts were cultured for an additional period of 48 hours in the presence of increasing doses of IL-2, as indicated, and in the presence (+NOK-1) or absence (Control) of 1 μg/mL of the anti-FasL–blocking mAb NOK-1. (B) Day-6 T-cell blasts were cultured for 24 or 48 hours, as indicated, in the absence (−) or presence of 100 ng/mL of the anti-Fas mAb CH-11 (CH11). (C) Day-6 T-cell blasts were cultured for 8 hours in the absence or presence of 100 ng/mL of the anti-Fas mAb CH-11, alone (CH11) or in combination with 1 μg/mL cycloheximide (CH11+CHX). After all the incubations described, cell extracts were obtained and anti-Bim or anti–β-actin immunoblots were performed. In panels B and C, extracts from day-6 T-cell blasts were also analyzed for comparison (noted as “6”). The extracts used correspond to 1 × 10⁶ cells, and the expression level of Bim_EL was quantified in a densitometer and normalized to the same amount of β-actin. The results obtained for the Bim_EL/β-actin ratios are shown in panels A and B. Results are representative of experiments performed with T-cell blasts from at least 4 different healthy donors. The samples shown in each immunoblot panel were run in the same gel. The vertical lines inside the subpanels in panel B indicate that lanes were cut from the same immunoblot membrane.