Figure 3.

Plasma membrane integrated hFWE4 assumes a four transmembrane pass structure with cytosolic N and C termini. Labeling of surface-exposed epitopes (A; left panels, B; left panels) in HEK293 cells stably coexpressing non-fused EGFP and (A) hFWE3 or (B) hFWE4 harboring epitope tags at indicated positions was performed by pulsing cells with the primary antibody on ice for 45 min prior to fixation and secondary antibody labeling. Labeling of total ectopic hFWE (A; right panels, B; right panels) was performed by fixing and permeabilizing cells prior to primary incubation. Shown are representative 100× confocal maximum intensity projections of immunolabeled hFWE (magenta), native EGFP (green), and Hoechst (blue), scale bar = 10 μm. C, schematic of hypothesized hFWE4 plasma membrane structure which presents N-terminal tail, L2, and C-terminal tail cytosolically, while L1 and L3 are exposed to the cell surface. hFWE3 is absent from the plasma membrane. Schematic shows hFWE3 orientation on intracellular membranes.