Fig. 1.

Necroptosis is activated by hyperphosphorylated tau in HT22 cells. A, B HT-22 cells were transfected with vector or TauP301S for 48 h. Nec-1 (30 μM) was added to the medium when the transfection medium was changed. A LDH was measured to assess cell death. B Cell death was analyzed by flow cytometry using Annexin V/PI staining. C Representative images of HT22 cells transfected with vector or TauP301S following treatment with DMSO or zVAD (30 μM) or zVAD (30 μM) + Nec-1 (30 μM) for 24 h, measured using Hoechst 33,258/PI staining, Scale bars, 100 μm; cell death was quantified by measuring LDH. Data are presented as the mean ± standard error of the mean (SEM) of three experiments, and a two-tailed unpaired t test was used to analyze the statistical significance of the data. D Cells were collected 48 h after transfection, and the lysates were analyzed by western blotting with the indicated antibodies. E HT22 cells were transfected with vector or TauP301S for 48 h. Cell lysates were immunoprecipitated with RIPK1 antibody, and the immunoprecipitated complexes were immunoblotted. F Immunoprecipitation and western blot analysis representative of three independent experiments. HEK 293 T cells were transfected with vector or TauP301S for 48 h. Cell lysates were immunoprecipitated with Flag beads, and the immunoprecipitated complexes were immunoblotted