Figure 2. Morphine and fentanyl administration lead to distinct MOR signaling complexes in striatum. a–d, Mice were treated with saline (sal), morphine (mor; 20 mg/kg), or fentanyl (fent; 0.125 mg/kg) for 30 min. Striatal extracts were IP with anti-MOR (a, b), β-arrestin-2 (c), or RGS9-2 (d) antibodies, and the immunoprecipitate was immunoblotted (WB) with the indicated antibodies. a, PLCβ3 was coimmunoprecipitated with MOR after fentanyl administration. b, Gαi3 was uniquely coimmunoprecipitated with MOR after morphine and showed low levels of interaction with MOR under baseline conditions or after fentanyl treatment. Data are expressed as means ± SEM. *p < 0.01 between treatments, one-way ANOVA followed by Dunnett's post hoc test. In c, striatal extracts were immunoprecipitated (IP) with an anti-β-arrestin-2 antibody, and the immunoprecipitate was immunoblotted (WB) for MOR. Activation of MOR by fentanyl or morphine increases its association with β-arrestin-2 (b; *p < 0.001 for morphine and fentanyl vs saline, **p < 0.05 for morphine vs fentanyl treatment, one-way ANOVA followed by Dunnett's post hoc test). Finally, d shows that MOR is immunoprecipitated with RGS9-2, and this interaction is strengthened after fentanyl or morphine application. Data are expressed as means ± SEM. *p < 0.01 between treatments, one-way ANOVA followed by Dunnett's post hoc test. For all experiments, n = 4–5 per treatment group. Ctr (control), Striata from morphine-treated mice immunoprecipitated with an anti-flag antiserum. WB for protein levels in total lysates are shown below each IP blot.