Figure 1: Characterization of palmitoylation-mutant Fas.
(a) Schematic of the Fas receptor, showing the location of palmitoylation site Cys199 (C194 in mouse). CRD: Cysteine-rich domain. PLAD: Pre-ligand assembly domain. (b) COS-7 cells were transfected with mEos2-tagged full-length WT, C199V Fas or WT Fas receptor lacking the death domain (ΔDD) and treated with crosslinked anti-Fas antibody. Individual cells were analysed via super-resolution PALM imaging. Minimum three cells were imaged (N=3–6) by PALM, with data representative from a single cell per treatment. Composite images were generated by combining single molecules localized in a PALM time series (3,000–5,000 images total). (c) HEK293T cells transfected with a truncated FADD (death domain only; FADD DD) and WT or C199V Fas receptor, either full-length or lacking the death domain (ΔDD), were analysed for interactions by FRET-based flow cytometry. Results are representative of two independent experiments. (d) HEK293T cells were transfected with a plasmid coding for a cyan-fluorescent protein (CFP) modified by signals for myristoylation and palmitoylation (MyrPalm), which targets CFP to lipid rafts65, as well as constructs containing WT or palmitoylation-deficient Fas C199V. Immunoblot analysis for Fas distribution was performed on Optiprep step gradient fractions of cell lysates 48 h post-transfection. WT or mutant C199V Fas-transfected HEK293T cells were incubated in the presence or absence of azide-linked palmitic acid and lysates were subjected to biotin-amide click chemistry. Immunoprecipitation of lysates was performed using streptavidin agarose beads and analysed for Fas by immunoblotting. Data are representative of three independent experiments.
