(A) Schematic illustration of topological inversion of C-terminally tagged GPCRs. (B) HEK-293 cells transfected with a plasmid encoding the indicated GPCR fused with a SNAP-tag at the C-terminus were treated with 8 μM C6-ceramide for 16 hr, and labeled with a cell permeable or impermeable SNAP-tag substrate. The ratio of fluorescent signal generated from cell impermeable versus that from cell permeable substrate was reported, with the value from cells untreated with C6-ceramide set at 1. Results were reported as Mean ±S.E. from triplicate incubations of a typical experiment. Similar results were obtained from two other independent experiments. (C–F) SV589 cells transfected with pCCR5-Myc were treated with C6-ceramide as described in B, and subjected to immunofluorescent microscopy analysis with anti-Myc in the absence (C, D) or presence (E, F) of saponin-mediated cell permeabilization. Scale bar = 50 μm.