Luciferase assay

The region of the wild-type PICK1 mRNA 3′UTR with a putative miR-615-3p binding site or mutant PICK1 mRNA 3′UTR were cloned into pMIR-REPORT Luciferase vector (cat # AM5795, Applied Biosystems) using Spe I and Hind III sites. The sequences of the putative binding site and the regions targeted by mutagenesis and cloned into the reporter gene. All plasmids were verified by sequencing. These constructs were transfected into indicated cells using Lipofectamine LTX with Plus Reagent (cat #18324–012, Life Technologies). Cells were plated at a density of 3600/cm² {(1 × 10⁴) per well, into a 96-well plate and attached overnight. They were co-transfected with 100 ng of wild-type or mutant reporter vector, 10 ng of internal control pRL-TK-Renilla-luciferase plasmid (cat# E2241, Promega) and negative control #1 or mirvana microRNA miR-615-3p mimic, both from Life Technologies final concentration, 80 nM. Twenty- four hours post-transfection, luciferase activities were measured using the Dual-Glo Luciferase Assay System (cat # E2920, Promega) according to the manufacturer’s instructions. Firefly luciferase values were normalized by dividing by the Renilla luciferase values.