2.5. RNA electrophoretic mobility shift assays (EMSA)

All oligonucleotides and primers used in this study were obtained from Integrated DNA Technologies (Coraville, IA). The oligonucleotides hsa-miR-25-3p: 5′-CAU UGC ACU UGU CUC GGU CUG A-3′ and hsa-miR-504-5p: 5′-AGA CCC UGG UCU GCA CUC UAU C-3′ were synthesized and 5′-labeled with cy5.5^TM dye. The 2′-O-methyl-modified RNA oligonucleotides CYP2B6-miR-25: 5′-CCA GGC UGG AGU GCU AUG GUG CAA UU-3′ and CYP2B6-miR-504: 5′-GGG GGU CAA AGG AUU CCA GGG UCA-3′, corresponding to the hsa-miR-25-3p and hsa-miR-504-5p targeting sequences resident in 3′-UTR of CYP2B6, were 5′-labeled with IRDye®800 dye (IDT). The unlabeled oligonucleotides, including the miRNA negative control (5′-UCA CAA CCU CCU AGA AAG AGU AGA-3′), hsa-miR-25-3p and hsa-miR-504-5p, were used in competition assays.

Cytoplasmic extracts were prepared from HepaRG cells using NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific). We performed the RNA EMSAs according to the manufacturer’s instructions (Thermo Scientific). Briefly, 1× RNA EMSA binding buffer, 5% glycerol, 200 mM KCl, 100 mM MgCl₂, and 200 nmol synthetic miRNA or/and cognate mRNA oligonucleotides were mixed in 20 μL reactions. Cytoplasmic extract (2 μg) and non-specific tRNA (1 μg, included in the RNA EMSA kit) were incubated 20 min at 4 °C to allow RNA:protein interactions to develop. Subsequently, antibodies against Ago1, Ago2, Ago3, and Ago4 (Abcam, Cambridge, MA) were used in the super-shift assays. In addition, unlabeled probes at 50-fold molar excesses were added to the reaction before the addition of dye-labeled probes in competition assays. The reaction mixtures were separated on a 12% PAGE by electrophoresis at 4 °C and detected with an Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).