Neuronal Cell Line Transfection and Culture

CAD (mouse catecholaminergic neuronal cell line, Cath.a-differentiated) cells were kindly given by Hubert Laude (Institut National de la Recherche Agronomique, Jouy-en-Josas, France) and cultured at 37 °C in Gibco Opti-MEM (Invitrogen), plus 10% fetal bovine serum and 1% penicillin/streptomycin^{23 (link)}. SH-SY5Y cells (neuroblasts from human neural tissue) were cultured at 37 °C in RPMI-1640 (Euroclone), plus 10% fetal bovine serum and 1% penicillin/streptomycin. Transient transfections were performed with Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. CAD cells were transfected in T25 flasks with the appropriate constructs: GFP-tagged constructs, GFP-VASP, GFP-Myo10, or H2B-mCherry for 3 h in serum-free medium and incubated O/N with complete medium. The following day, transfected cells were plated on grids^{19 (link)}.