Palmitate Labeling and Nmnat2 Transfection

HEK 293 cells were maintained in culture, transfected and labeled with [9,10-³H]palmitate (PerkinElmer Life Sciences) as described (11 (link)). Where indicated, 50 μm PSB was added at the same time as the radiolabeled palmitate or 5 h after addition of palmitate (i.e. 1 h before cell lysis). HA (50 mm) was added for 60 min prior to cell lysis (i.e. 5 h after addition of radiolabel). Neuroblastoma X spinal cord cells (NSC34) were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Sigma), 1% penicillin/streptomycin (Invitrogen), 2 mml-glutamine (Invitrogen), and 1 mm sodium pyruvate (Invitrogen). NSC34 cells in six-well dishes were transfected with 4 μg of FLAG-Nmnat2 and 1.35 μg of each of the indicated mouse siRNA construct(s) (Thermo Scientific) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Seventy-two hours after transfection, palmitate labeling was carried out as for HEK 293 cells.