Generating Stable Cell Lines Expressing HA-TDP-43

Generation of HEK293^HA-TDP-43 stable cell lines, expressing HA-tagged WT, E17R, S48A, S48E, M337V, and ΔCR constructs and stable cell lines was previously described [40 (link)]. Mutants A326P and M337P were generated by site-directed mutagenesis as previously described [10 (link)] using the primers listed in S1 Table. Briefly, the HEK293-derived cells Flp-In T-Rex 293 Cell Line (Thermo Fisher Scientific) were stably transfected to express HA-TDP-43 upon induction with tetracycline (1 μg/ml). Cells were grown and maintained in DMEM (Dulbecco’s Modified Eagle’s Medium–High Glucose, Corning) supplemented with 10% FBS (fetal bovine serum) and incubated in a humid atmosphere at 37°C and 5% CO₂. Expression of HA-tagged TDP-43 construct was induced at 30% confluence.