Fig. 2. (a) Map of FasL cytoplasmic tail showing mutations made during this study. The amino acid sequence of human FasL cytoplasmic tail. FasL is a type-II membrane protein and so the cytoplasmic tail represents the N terminus of the protein. The map outlines the deletions made to the tail during this study (37, 54, 67 and 74), the proline-rich domain, the two putative SH3-domain-binding motifs (arrows underneath denote the orientation), the KKR and RR residues (boxed), orientation of the GFP tag in the FasL-GFP construct, and the beginning of the transmembrane (tm) region of FasL. The first (M1) and last (G80) amino acid of the tail are numbered above the sequence. (b-d) Progressive N-terminal deletion of the cytoplasmic tail of FasL results in increased cell surface expression. FasL37-GFP, FasL54-GFP, FasL67-GFP and FasL74-GFP and FasLWT-GFP were transiently expressed in RBL cells and FasL cell surface expression was detected by confocal microscopy (b) and FACS analysis (c,d). For confocal analysis, the transfectants were fixed in ice-cold methanol 36 hours post-transfection and stained with Nok-1 antibody, which recognises the extracellular domain of FasL. The GFP and Nok-1 signals showed complete overlap; the images shown are from the Nok-1 signal. Unpermeabilised cells for FACS analysis were stained with Nok-1 as a measurement for membrane expression, and the total GFP signal was used as a measurement for total FasL. Double plots of GFP vs Nok-1 stain are shown (c). For each transfectant, the Nok-1 stain for cells expressing between 10 units and 150 units of GFP fluorescence (shown gated in c) was plotted (d). The percentage of cell surface FasL is shown in the top right-hand corner of the FACS plot.