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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 2.

Figure 2

AM3677 engages hCB1R covalently at TMH6 cysteine residue C6.47(355). (A) Preincubation of Flp-In-293 membranes from cells expressing hCB1R with excess (10-fold its apparent Ki) AM3677 reduces subsequent [3H]CP55,940 specific binding (i.e., Bmax) by at least 43% to hCB1Rs containing C6.47(355) [as illustrated for WT hCB1R and the C4.47(238)S receptor mutant]. In contrast, the saturation-binding profile of [3H]CP55,940 is unaffected in the hCB1R C6.47(355)S mutant. (B) Comparison of the extent of covalent AM3677 labeling of WT hCB1R and mutants, designated as the difference in the respective [3H]CP55,940 Bmax values of each hCB1R membrane preparation with or without preincubation with AM3677. Data shown represent the mean ± SEM of three independent experiments performed in duplicate.