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. Author manuscript; available in PMC: 2016 Mar 23.
Published in final edited form as: ACS Chem Neurosci. 2015 May 29;6(8):1400–1410. doi: 10.1021/acschemneuro.5b00090

Figure 5.

Figure 5

(A) AM3677 internalizes CB1R in a concentration-dependent manner. HEK293 cells expressing hemagglutinin-tagged rCB1R were incubated with each indicated concentration of AM3677 for 30 min at 37 °C. Loss of cell-surface receptors was quantified as detailed in the text, and a curve of best fit was plotted by nonlinear regression. Values shown are from a single experiment performed in triplicate. (B) AM3677-induced CB1R internalization is irreversible. Treatment for 2 h with AM3677 (300 nM) or CP55,940 (10 nM) induced rCB1R internalization, designated as the relative percent vehicle control cell-surface expression level. Subsequent exposure to CP55,940/SR141716A (SR) (100 nM final SR141716A concentration) resulted in recycling of internalized CB1Rs to the plasma membrane, as demonstrated by the recovery of cell-surface CB1R immunoreactivity after 30 and 60 min. In contrast, CB1R recycling was not observed following AM3677/SR141716A treatment. Values shown are from three independent experiments done in quadruplicate (** p < 0.01; * p < 0.05; ns, not significant. One-way ANOVA with Dunnett’s post-test, as compared to respective vehicle-treated control).