FIGURE 9.

Orexin A-induced internalization of CB₁-OX₁ heteromer. Clone B6 cells were uninduced (lanes 1 and 6) or induced with doxycycline (10 ng·ml⁻¹ for 24 h) (all other lanes). Following cell surface biotinylation, cells were treated with vehicle (lanes 2 and 7) or either orexin A (OxA) (lanes 3–5) or WIN55212-2 (lanes 8–10) (10⁻⁶ m (lanes 3 and 8), 10⁻⁷ m (lanes 4 and 9), or 10⁻⁸ m (lanes 5 and 10)) for 40 min. Biotin was cleaved from proteins remaining at the cell surface, and the internalized and therefore protected CB₁ receptor was detected following SDS-PAGE. A representative experiment of n = 3 is shown in A. Individual blots were scanned, and polypeptides were quantified and normalized. Combined results are displayed as means ± S.E. in B. VS, VSV-G + SNAP tag; HC, HA + CLIP tag. Polypeptides with apparent mass labeled (i–iv) in A were quantitated in B across lanes 1–10.