Cell culture, transfection, and treatments

NG108-15 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% dialyzed fetal bovine serum (FBS), 2% hypoxanthine/aminopterin/thymidine (Life Technologies), and antibiotics. Cells were transfected in suspension using Lipofectamine 2000 (Life Technologies) with plasmids encoding GFP, WT, or mutant HA–5-HT₆Rs with or without the plasmid encoding GPRIN1. After transfection, cells were maintained in DMEM supplemented with 2% dialyzed FBS and treated as indicated in the figure legends either 5 or 24 hours after transfection. The cells were used either 24 or 48 hours after transfection for all experiments. Primary cultures of striatal neurons were prepared from E15.5 OF1 mouse embryos. Striata were dissociated by manual trituration, and cells were plated onto polyornithine- and laminin-coated 12-mm glass coverslips in 24-well plates. Cultures were maintained in Neurobasal medium (Thermo Fisher Scientific) supplemented with 10 ml of B27 supplement (Gibco, ref 17504-044), 0.2 mM GlutaMAX, with 0.2 mM glutamine and penicillin (100 U)/streptomycin (100 μg) and kept at 37°C in a humidified atmosphere containing 5% CO₂. For branching experiments, neurons were transfected at DIV 1 with cDNA encoding GFP to visualize dendritic trees, treated at DIV 4 as indicated in the figure legends, and fixed at DIV 13.