DNA transfection of HT22 cells and primary cultures to perform luciferase assay

HT22 cells were seeded at 15 × 10³ cells per well in 96-well plates. Afterwards, a total of 50 ng DNA/well (25 ng Renilla luciferase + 25 ng CMV-luciferase vectors; 25 ng Renilla luciferase + 25 ng GluN2B-5′UTR CMV-luciferase vectors; 25 ng Renilla luciferase + 25 ng GluN2B-5′UTR triple mutant CMV-luciferase constructs) was transfected using the JetPEI transfection reagent (PolyPlus, Korea). The medium was replaced after 4 h and treatments were carried out at 48 h to allow sufficient gene expression. For primary hippocampal cultures, 50 × 10⁴ cells per well were seeded in 6-well plates, and at DIV8 were transfected with a total of 4 µg of DNA per well with NeuroMag (described above). At DIV10 neurons were incubated with Wnt5a medium for 1 h, and then lysed to measure luciferase/Renilla activities following the manufacturer’s instructions for the Dual-Glo Luciferase Assay System (Promega). Luminescence was read using a luminescence reader (Turner Designs Luminometer Model TD20/20).