Quantitative Western Blotting Analysis

Western blotting analysis was performed as previously reported^{62 (link)}. An equal amount (40–80 μg) of protein samples were detected. Primary antibodies against p-MLKL (Rabbit, 1:500, ab187091; Abcam, UK), MLKL (Rabbit 1:1000, ab194699; Abcam, UK), p-RIP1 (Rabbit, 1:500, 65746; Cell Signaling Technology, USA), RIP1 (Mouse, 1:500, ab72139; Abcam, UK), caspase 8 (Rabbit, 1:1000, A0215; ABclonal, China), caspase 3 (Rabbit, 1:1000, A11319; ABclonal, China), TNF-α (Rabbit, 1:500, ab6671; Abcam, UK), IL-6 (Mouse, 1:500, ab9324; Abcam, UK), iNOS (Rabbit, 1:500, ab15323; Abcam, UK), IL-1β (Rabbit, 1:500, ab2105; Abcam, UK), NF-kB p65 (Rabbit, 1:1000; ab16502, Abcam, UK), IkBα (Mouse, 1:1000, 4814; Cell Signaling Technology, USA), p-IkBα (Rabbit, 1:1000, 2859; Cell Signaling Technology, USA); H3 (Rabbit, 1:1000, AF0009; Beyotime, China) and β-actin (Rabbit, 1:3000, AC026; ABclonal, China) were used. Protein expression levels were analyzed using ImageJ software and normalized to β-actin (National Institutes of Health, Bethesda, MD, USA). Phosphorylated protein expression was evaluated compared to total protein expression.

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Chen A.Q., Fang Z., Chen X.L., Yang S., Zhou Y.F., Mao L., Xia Y.P., Jin H.J., Li Y.N., You M.F., Wang X.X., Lei H., He Q.W, & Hu B. (2019). Microglia-derived TNF-α mediates endothelial necroptosis aggravating blood brain–barrier disruption after ischemic stroke. Cell Death & Disease, 10(7), 487.

Publication 2019

ab187091 ab194699 ab72139 A11319 ab6671 ab9324 ab15323 ab2105 ab16502 p-IkBα AF0009 AC026

Actin Antibodies Caspase 3 Caspase 8 Il 1β Inos Mouse Nf kb Protein Rabbit Tnf α Western blotting analysis

Corresponding Organization :

Other organizations : Union Hospital, Huazhong University of Science and Technology, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences

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Variable analysis

independent variables

Protein samples (40-80 μg)

dependent variables

Protein expression levels of p-MLKL, MLKL, p-RIP1, RIP1, caspase 8, caspase 3, TNF-α, IL-6, iNOS, IL-1β, NF-kB p65, IkBα, p-IkBα

control variables

H3 protein expression
β-actin protein expression

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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