JNK and ERK are not the main pathways that control Bim levels in human T-cell blasts. (A) Day-6 T-cell blasts were cultured for an additional period of 48 hours in the presence of 30 IU/mL of IL-2 and in the presence (+NOK-1) or absence (−) of 1 μg/mL of the anti-FasL–blocking mAb NOK-1. (B) Day-6 T-cell blasts, obtained from healthy donors (Control) or from ALPS-Ia patient 2 or ALPS-Ic patient 1, as indicated, were analyzed without further incubation. (C) Day-6 T-cell blasts from healthy donors were cultured for an additional period of 24 hours in the presence of 30 IU/mL of IL-2 and in the presence of either 0.2% DMSO (Control) or 10 μM PD098059, as indicated. After the incubations described, cell extracts were obtained and anti–phospho-JNK1/2 (P-JNK1/2; A), anti-Bim (A,C), anti–phospho-ERK1/2 (PERK1/2; B-C), or anti–β-actin (A-C) immunoblots were performed. The extracts used correspond to 1 × 10⁶ cells, and the levels of P-ERK1 or P-ERK2 were quantified in a densitometer and normalized to the same amount of β-actin (not shown). Results are representative of experiments performed with T-cell blasts from 3 different healthy donors. The samples shown in each immunoblot panel were run in the same gel. The vertical lines inside the top subpanels in panel B indicate that lanes were cut from the same immunoblot membrane.