Origin 5

In vitro binding, [³⁵S]GTPγS stimulation, and β-arrestin-2 coupling experiments were analyzed using Origin 5.0 software (OriginLab), with sigmoidal fitting to determine parameters of half-maximal concentrations and maximal intrinsic activity. In the case of binding experiments, sigmoidal IC₅₀ determinations were converted to K_i values using the Cheng-Prusoff equation (Yung-Chi and Prusoff, 1973). The K_d values used were obtained from saturation binding analyses for [³H]U69,593, [³H]DAMGO, and [³H]DPDPE for the respective KOR, MOR, and DOR cell lines, using Scatchard analysis. For intrinsic activity calculations, values were normalized to that obtained with concurrent reference ligand stimulation. The reference ligand used for all in vitro assays was U69,593. All in vitro determinations were conducted in a minimum of 3 separate experiments In vivo rotarod and prolactin experiments were analyzed using Statistica 13.0 statistical software (Dell Statistica). For rotarod experiments, 2-way ANOVAs with repeated measures (condition/dose x time, with repeated measures on time) were used to examine effects and/or interactions of condition/dose and time. For prolactin experiments, 1-way ANOVAs were used to examine effect of condition. In both cases, Newman-Keuls posthoc tests were utilized to examine significant differences.

Total RNA was prepared using TRIzol^® reagent (Invitrogen). For miRNA quantification, cDNA was synthesized with specific miRNA reverse transcriptase primers using the TaqMan microRNA Reverse Transcription kit (Applied Biosystems). For mRNA quantification, cDNA was reversely transcribed at 37°C for 15 min, and 95°C for 10 min using the Moloney murine leukemia virus (M-MLV) reverse transcriptase (Toyobo Life Science), according to the manufacturer's protocol. RNA and cDNA samples were stored at −80°C until use. The qPCR analysis was performed on an Exicyler 96 sequence detection system (Bioneer Corporation). The final 50 µl reaction mixture contained 25 µl SYBR-Green PCR Master Mix (Toyobo), 2 µl primer mix (5 µM) and 10 ng cDNA. Thermocycling was carried out as follows: 95°C for 2 min for denaturation; followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec for amplification; and 72°C for 30 sec for termination. The standard curve was plotted using Origin 5.0 software (Originlab Corporation). The relative expression of miRNA was calculated using the 2^−ΔΔCq method (17 (link)) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following primers for GAPDH were synthesized by Beijing Dingguo Changsheng Biotechnology Co., Ltd.: Sense, 5′-CCATGGAGAAGGCTGGGG-3′ and antisense, 5′-CAAAGTTGTCATGGATGACC-3′.