Expression of the short and long forms of FLIP in resting and activated NK cells, CD8⁺ T cells, and in the NK 3.3 cell line. (A) RT-PCR of FLIP_Short and FLIP_Long in resting and activated (as described in “Materials and methods”) NK and CD8⁺ T cells for 1 day or 10 days. MCF7 cells served as positive controls. (B) RT-PCR of FLIP_Short and FLIP_Long in CD8⁺ T cells activated by anti-CD3, IL-2 alone, or PHA alone. The expression of FLIP mRNA is not dependent on the type of stimulation used to activate CD8⁺ CTLs. (C) Western blot analysis of FLIP protein in resting (0 day) and stimulated (10 days) NK cells, CD8⁺ T cells, and NK 3.3 cell line. (D) Quantification of gene transcription by semiquantitative RT-PCR in NK cells before and after IL-2 stimulation (lanes 1-5, 1/8, 1/20, 1/100, 1/500, 1/2500 cDNA dilutions were analyzed to detect FLIP_Short and FLIP_Long; lane 6, RT-PCR negative control). (E) RT-PCR analysis of FLIP_Short and FLIP_Long expression with or without actinomycin D treatment (lanes 1 and 4, β-actin; lanes 2 and 5, FLIP_Short; lanes 3 and 6, FLIP_Long RT-PCR products). (F) Flow cytometry analysis of the surface expression of TRAIL receptors in NK cells with or without actinomycin D treatment (50 ng/mL for 24 hours). Empty histograms indicate controls, untreated cells. Gray histograms indicate actinomycin-treated cells.