Comparison of hMOR and Ala348,353 hMOR [35S] GTPγS binding with or without PTX treatment. Basal (panel A) and DAMGO-induced (panel B) [35S] GTPγS incorporation were measured as described in the Experimental Procedures. Membranes were prepared from HEK 293 cells stably expressing hMOR or Ala348,353 hMOR. Cells were treated with 100 ng/mL PTX for 20 h where indicated. DAMGO was added at a concentration of 10 μM. Data are given as the mean ± S.E.M. from at least 3 independent experiments performed in triplicate. Statistical analysis was performed using Student's t test to evaluate the effects of mutation (***, p < 0.001) or PTX-treatment (##, p < 0.01) on basal [35S] GTPγS incorporation (panel A) and of the effect of PTX-treatment (**, p < 0.01) on DAMGO-induced [35S] GTPγS incorporation (panel B).