Lenti vpak

We used HEK293T cells for immunoprecipitation assays and the NSC34 cell line as a motoneuron-cell like a model [41 (link)] for autophagy experiments, subcellular localization studies and mutant SOD1 aggregation and cell death analysis. NSC34 and HEK 293 T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 12,800,017) supplemented with 10% fetal bovine serum (FBS, Gibco, 10,438,026) and 1% penicillin/streptomycin (Biological Industries, DW1012), in a 5% CO₂ incubator at 37 °C. All transfections were performed using the Effectene reagent (Qiagen, 301,427) according to the manufacturer’s recommendations. Plasmid DNAs were prepared using the Qiagen plasmid midi kit (Qiagen, 12,143) or the Axygen miniprep kit (Axygen, AP-MN-P-250). For viral transductions, lentivirus production was performed with Lenti-vpak (Origene, TR30037), according to the manufacturer’s recommendation. Briefly, HEK293T cells were transfected with scramble control (shCtrl) and shPacer constructs. Medium was replaced with fresh DMEM after 12 h, and viral supernatant was collected after 36 h and again after another 24 h. NSC34 cells were transduced with 2 ml viral supernatant. Stable NSC34 shCtrl and shPacer cell lines were established with Puromycin (Sigma, P8833-25 mg) (10 μg/ml) selection.