Dapk1 inhibitor

The Escherichia coli 0111: B4 LPS, N-hexanoyl-D-sphingosine (C6-ceramide) and zVAD-FMK were obtained from Sigma-Aldrich (St. Louis, MO, USA). DAPK1 inhibitor and ST2825 were purchased from Medchem Express (Monmouth Junction, New Jersey, USA). Recombinant GST-DAPK1 fusion protein was obtained from Millipore (Billerica, MA). Caspase-3 activity detection kit was from Bestbio (Shanghai, China). Quantikine human IL-6 ELISA kit was from R&D Systems (Minneapolis, MN). Annexin V-FITC apoptosis detection kit was ordered from Beyotime (Nanjing, China). The following antibodies with the company and concentration were used for coimmunoprecipitation (co-IP) or western-blotting analyses: anti-Pellino1 (Abcam, 1:500), anti-MyD88 (Cell Signaling Technology, 1:500), anti-caspase-8 (Cell Signaling Technology, 1:500), anti-TRIF (Cell Signaling Technology, 1:1000), anti-RIP1 (BD Biosciences, 1:2000), anti-Flag (ProteinTech group, 1:1000), anti-phospho-DAPK1 (Sigma-Aldrich, 1:1000), anti-DAPK1 (Cell Signaling Technology, 1:1000), anti-Fbxw7 (Abcam, 1:1000), anti-pSer (Santa Cruz, 1:1000), anti-Fn14 (Cell Signaling Technology, 1:2000) and anti-GAPDH (Biosynthesis, 1:3000).

Bv2 cells, a murine microglial cell line, were obtained from Cell Resource Center of Peking Union Medical College (Beijing, China), and maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (all from Gibco, Grand Island, NY, USA) in a humidified incubator with 5% CO₂ at 37 °C.
We primed cells with ultrapure lipopolysaccharide (LPS, 100 ng/ml) (Sigma-Aldrich, St. Louis, MO, USA) for 6 h and washed with PBS. After that, cells were further stimulated with Aβ_25–35 (25 μM) or fibrillar Aβ_1–42 (10 μM) or oligomeric Aβ_1–42 (10 μM) for varying times (6–48 h). In some experiments, LPS-primed cells were incubated with cathepsin B inhibitor CA-074Me (1, 2.5, 5 μM; Calbiochem, Darmstadt, Germany) or DAPK1 inhibitor (2.5, 5, 10 μM; MedChem Express, Monmouth Junction, NJ, USA)) or for 1 h prior to Aβ_25–35 treatment.