Lentiviral transductions and stable cell lines

A stable cell line expressing mCherry-Rab35 was obtained by transfection using AMAXA, followed by selection with 100 ng μl⁻¹ Genetecin (G418) for 2 weeks and FACS sorting for mCherry-positive cells. Stable cell lines expressing control shRNA IRES GFP, Rab35 shRNA IRES GFP, control shRNA or Rab35 shRNA are obtained by lentiviral transduction (10 multiplicity of infection, 10⁷ particles per ml) in growth medium and 5 μg ml⁻¹ Polybrene, following selection with 1 μg ml⁻¹ Pyromycin 72 h after transduction. Cells expressing control shRNA IRES GFP or Rab35 shRNA IRES GFP were sorted by FACS for low, medium and high levels of GFP fluorescence intensities.