Neuroblastoma x Spinal cord hybrid NSC-34 cells69 (link) were maintained in Dulbecco’s modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum (FBS, Bovogen Biologicals, Australia). Cells were kept at 37 °C in a humidified incubator with 5% atmospheric CO2. For confocal microscopy, cells were grown on 13 mm round coverslips in 24-well plates or on 4 or 8-well chambered coverglass (Nalge Nunc International, USA). Cells were grown in 6-well plates for cell lysate experiments.
Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) according to manufacturer’s instructions with 0.5 μg DNA per well for a 24-well plate or 4-well chambered coverglass, 0.2 μg DNA per well for 8-well chambered coverglass and 2 μg DNA per well for 6-well plates. For co-transfections and triple transfections, the amount of DNA was divided equally between constructs. The DNA: Lipofectamine ratio used was 1:5 (w/v). Media was refreshed 5 h after transfection. For disruption of microtubules, cells were incubated with 33 μM nocadazole (Sigma-Aldrich, USA) overnight (~ 18 h) before analysis 48 h post-transfection.
Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) according to manufacturer’s instructions with 0.5 μg DNA per well for a 24-well plate or 4-well chambered coverglass, 0.2 μg DNA per well for 8-well chambered coverglass and 2 μg DNA per well for 6-well plates. For co-transfections and triple transfections, the amount of DNA was divided equally between constructs. The DNA: Lipofectamine ratio used was 1:5 (w/v). Media was refreshed 5 h after transfection. For disruption of microtubules, cells were incubated with 33 μM nocadazole (Sigma-Aldrich, USA) overnight (~ 18 h) before analysis 48 h post-transfection.