Enhanced mRNA translation of c-FLIP_S over c-FLIP_R. (A) GFP-tagged c-FLIP_S or c-FLIP_R was transiently overexpressed in 293T cells. Differences in protein expression were determined by evaluating the GFP fluorescence intensity via FACS analysis. For relative quantification of low (M1), medium (M2), and high (M3) GFP-c-FLIP expression levels gates were set as indicated. (B) HeLa cells were transiently transfected with either GFP-c-FLIP_S or GFP-c-FLIP_R; 24 hours after transfection, the cells were fixed and stained with DAPI (blue). Subsequently, samples were analyzed by confocal laser scanning microscopy. (C left panel) Coupled in vitro transcription/translation of c-FLIP_S and c-FLIP_R. 5, 2, or 1 μL of translated proteins were separated via SDS-PAGE and analyzed by Western blotting. The second (bottom) c-FLIP bands are probably caused by translational start at an internal methionine. (C right panel) c-FLIP_S or c-FLIP_R were in vitro transcribed and then equal RNA amounts were applied for an in vitro translation assay. Protein levels of the 2 c-FLIP isoforms were determined by Western blot.