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Figure S1 SKNSH cells (human neuroblastoma) treated with (±)-U-50488 hydrochloride (a κ agonist). SKNSH cells (106 cells per well) endogenously expressing κ-receptors were treated with the indicated concentrations of (±)-U-50488 hydrochloride. Cells were incubated overnight at 4°C with 1 μg per well of antireceptor antibodies, then washed three times with PBS and incubated with a secondary antibody (dilution 1:500) of alkaline phosphatase-conjugated anti-rabbit antibodies for 60 min at room temperature. After washing three times with PBS, the amount of binding was detected using phosphatase substrate. The extent of receptor recognition by the antibodies was assayed by elisa. Data from the vehicle-treated cells were taken as 100%. All of the results of the experiments are represented as the percentage of the control. The results are expressed as mean ± SEM (n = 3).

Figure S2 Rat spleen membranes treated with Δ9-THC. Rat spleen membranes (5 μg per well) were treated with 1 μM concentration of either Δ9-THC (CB2 agonist) or WIN 55212-3 (CB2 antagonist). Cells were incubated overnight at 4°C with 1 μg per well of antireceptor antibodies, washed three times with PBS and incubated with a secondary antibody (dilution 1:500) of alkaline phosphatase-conjugated anti-rabbit antibodies for 60 min at room temperature. After washing three times with PBS, the amount of binding was detected using phosphatase substrate. The extent of receptor recognition by the antibodies was assayed by elisa. Data from the vehicle-treated cells were taken as 100%. All of the results of the experiments are represented as the percentage of the control. The results are expressed as means ± SEM (n = 3).

Figure S3 In vivo assay for the κ-opioid receptor. Mice were injected with either saline or 10 mg·kg−1 of dynorphin A (i.p.) and killed 30 min after the injection. Brains were immediately collected and stored at −80°C until used. The immunostaining technique was performed in tissue slices from the mouse brain using conformation state-sensitive anti-κ antibodies. Primary antibodies were labelled with fluorescent dyes, and fluorescence was measured using Odyssey equipment from Li-Cor. (A) Images from brain sections representing striatum from the control and treated mice were probed with conformation-specific anti-κ-opioid receptor antibodies. (B) Data quantification from striatum-treated and control mice. Vehicle-treated mice were taken as 100%. The results are expressed as means ± SEM (n = 3).

Figure S4 HEK293 cells expressing the CB2 receptor treated with Δ9-THC. HEK293 cells expressing the CB2 receptor were treated with 1 μM concentration of agonist (Δ9 -THC). The cells were incubated overnight at 4°C with 1 μg per well of anti-receptor antibodies, washed three times with PBS and incubated with a secondary antibody (dilution 1:500) of alkaline phosphatase-conjugated anti-rabbit antibodies for 60 min at room temperature. After washing three times with PBS, the amount of binding was detected using phosphatase substrate. The extent of receptor recognition by the antibodies was assayed by elisa. Data from the vehicle-treated cells were taken as 100%. All of the results of the experiments are represented as a percentage of the control. The results are expressed as means ± SEM (n = 3).

Figure S5 Specificity of the conformation-sensitive receptor antibodies. Paw tissues from animals treated with either ACEA (CB1 agonist) or AM1241 (CB2 agonist) 2 h after PGE2 (100 ng per paw) injection were incubated with anti-CB1 and anti-CB2 conformation-sensitive receptor antibodies (Figure 5A). Paw tissues from animals treated with DAMGO (μ-agonist), U50.488 (κ-agonist) or DPDPE (δ agonist) 2 h after PGE2 (100 ng per paw) injection were incubated with anti-μ, anti-κ and anti-δ conformation-sensitive receptor antibodies (Figure 5B). The analysis and quantification of the fluorescence was performed using the Odyssey system (Li-Cor). The fluorescence of the naïve rats was taken as 100% (dashed line), and the fluorescence of the treated groups was represented as percentage of activation compared with the control group. This control group (negative control) represents slices treated only with secondary antibody labelled with fluorescent Alexa Fluor dyes (682 and 800 nm). The results are expressed as means ± SEM (n = 5 animals for control group and 6 animals for experimental group). *P < 0.05 compared with the control group measurement (dashed line).

Table S1 Cross-reactivity assay. SKNSH cells (human neuroblastoma) were treated with various agonists (1 μM). Cells were incubated overnight at 4°C with 1 μg per well of anti-receptor antibodies, washed three times with PBS and incubated with a secondary antibody (dilution 1:500) of alkaline phosphatase-conjugated anti-rabbit antibodies for 60 min at room temperature. After being washed three times with PBS, the amount of binding by the cells was detected by a phosphatase substrate. The extent of receptor recognition by the antibodies was assayed by elisa. Data from the vehicle-treated cells were taken as 100%. The results are represented as the percentage of the control (buffer treatment). Data from the vehicle-treated cells were taken as 100%. The results are expressed as means ± SD (n = 3).

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