Cultivation and Transfection of Kidney Cells

Opossum kidney cells (OKP cells) were maintained in DMEM (Gibco 42430) supplemented with 10% fetal bovine serum (Eurobio, Les Ulis, France), penicillin (100 U/mL), and streptomycin (100 U/mL) at 37 °C in a humidified atmosphere containing 5% CO₂. Human embryonic kidney (HEK) 293 cells were grown in DMEM media complemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For plasmid DNA transfection, cells were grown to 60–70% confluence before being transiently transfected for 5 h using Lipofectamine plus kit according to manufacturer’s instructions (Invitrogen, Paris, France). For protein degradation experiments, cells were treated with MG132 (2 μM) or chloroquine (100 μM) 6 h prior to cell lysis, as previously described [53 (link),61 (link)]. Kifunensine (Sigma k1140, Saint-Quentin-Fallavier, France) was used at 25 µM 6 h before cell lysis.