Cell cultures and treatments
Three cell lines were employed in this study: HEK-293, iHEK overexpressing WT tau and iHEK overexpressing mutated P301L tau. All three were maintained in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 oC in 5% CO2. To induce WT and mutant tau overexpression, iHEK cells were treated with 1 µg/ml tetracycline (Tet) for 24 h in FBS-depleted DMEM (GibcoTM LS11965118, Thermo Fisher Scientific). After 24 h incubation, cells were washed twice with medium to remove excess Tet, then collected and stained. Trypsin (GibcoTM Trypsin-EDTA (0.25%) phenol red (LS25200114 Thermo Fisher Scientific) was used added and incubated for 3 min to detach cells, and the solution was then centrifuged at 1000 × g for 5 min. Cell pellets were collected and used for protein fractioning.
Human tissue processing
Postmortem…
