Skip to main content
. 2010 Oct 13;30(41):13774–13783. doi: 10.1523/JNEUROSCI.0091-10.2010

Figure 5.

Figure 5.

REEP2 recruits T1R receptors to lipid rafts. A, Cell surface proteins from transfected HEK293E cells expressing mT1R2-FLAG and mT1R3-HA in the presence (rows 1, 3) or absence (rows 2, 4) of REEP2 were biotinylated, isolated, electrophoresed in polyacrylamide gels and transferred to PVDF membranes. Western blots probed with anti-FLAG, anti-HA, or anti-REEP2 antibodies showed that the presence of REEP2 did not increase cell surface expression of T1R2 or T1R3 receptors, nor did it affect their expression among total cell protein. B, Lipid raft proteins from transfected HEK293E cells expressing mT1R2-FLAG, mT1R3-HA and actin-EYFP in the presence (rows 1, 3) or absence (rows 2, 4) of REEP2 were isolated (see supplemental Fig. 6, available at www.jneurosci.org as supplemental material). Western blots probed with antibodies against FLAG and HA, and dot blots probed with antibodies against GFP, integrin-β1 and REEP2 showed that the presence of REEP2 increased accumulation of both T1R2 and T1R3 in lipid rafts. In contrast, expression of both sweet receptor subunits among total cellular protein was unaffected by the presence of REEP2. Expression of actin-EYFP serves as a normalization marker for each preparation and is independent on the presence or absence of REEP2. Note that actin filaments—known to be associated with lipid rafts (Bodin et al., 2005; Taguchi et al., 2005)—were also found in isolated lipid rafts, while the membrane protein integrin-β1—excluded from lipid rafts (Bodin et al., 2005)—was not. C, D, Projection of consecutive confocal images covering a 7 μm tissue section illustrates the distribution of REEP2 within the taste bud. D, Overlay of red and transmission channels indicates that REEP2 is more highly expressed near the taste bud pore region, suggesting a role in the spatial organization of taste receptors to the apical domain (arrows). E, REEP2 localization was quantified from the apical to the basal side of 7 distinct taste buds in 2-mm-thick longitudinal sections by immunostaining, showing the abundance of REEP2 proteins in proximity to the apical pore. REEP2 was detected using an anti-REEP2 antibody and indirect immunofluorescence (red). Representative data are presented. Scale bar, 10 μm.