Regulation of Tip110 expression by miR-124 through its 3′-UTR. (A) Sequence alignment between human miR-224 seed sequence and 3′-UTR of Tip110 mRNA from different species, including mouse, chimpanzee, rhesus, bushbaby, rat, and guinea pig. (B, C) 293T were transfected with miR-124 mimic (miR-124m) or its control (Ctrlm). Forty hours after transfection, cells were harvested for cell lysates and western blotting (B) or RNA isolation and real time polymerase chain reaction (qRT-PCR) (C). β-Actin was included as the loading control for western blotting (B) and as a reference for qRT-PCR (C). (D) Scheme of human Tip110 3′-UTR-containing luciferase reporter plasmid pLightSwitch-Tip110.3′UTR. (E, F) 293T were transfected with miR-124m and pLightSwitch-Tip110.3′UTR (E) or miR-124-binding site-deleted pSwitchgear-Tip110.3′UTR (F). Forty-eight hours after transfection, cells were harvested for cell lysates and luciferase reporter gene assay. pTK-βGal was included for transfection efficiency normalization (E, F). The data in (D, E) are mean±SEM and representative of three independent experiments. 3′-UTR, 3′-untranslated region.