Increased protein expression of c-FLIP_S. (A) L428 cells were treated for the indicated times with 10-μg/mL cycloheximide. c-FLIP expression was analyzed by Western blot. β-Actin served as a loading control. (B) L428 cells were treated for the indicated times with actinomycin D (ActD, 1 μg/mL). Endogenous c-FLIP isoform mRNA stability was analyzed by RT-PCR. (C) 293T cells were transiently transfected with the indicated cDNA amounts for c-FLIP_S or c-FLIP_R; 24 hours after transfection, cells were treated for up to 8 hours with 10 μg/mL cycloheximide. Protein stability of the 2 c-FLIP isoforms was monitored by Western blot analysis. Tubulin was analyzed to control equal protein loading. The weaker separation of c-FLIP_S and c-FLIP_R, compared with panel A, is due to different polyacrylamide concentrations of the gel. Additional faster migrating bands of c-FLIP_S and c-FLIP_R are presumably caused by translational start at an internal methionine and marked by an asterisk. (D) 293T cells were cotransfected with equal amounts of c-FLIP_S– and c-FLIP_R–encoding plasmids; 24 hours later, cells were stimulated for the indicated times with actinomycin D (1 μg/mL). mRNA stability was assessed by RT-PCR. The asterisk marks a cDNA band of unclear identity that appears specifically in the transfected cells.