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. 2019 Feb 19;6(1):ENEURO.0308-18.2018. doi: 10.1523/ENEURO.0308-18.2018

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Copyright © 2019 Dermentzaki et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 1. — Upregulation of core necroptosis components in the spinal cord of symptomatic Tg SOD1^G93Amice. Lumbar spinal cords, from 12- and 15-week-old Tg SOD1^G93A, WT SOD1, and NTg mice, were isolated and processed for mRNA and protein (RIPA or urea extraction) expression of RIPK1, RIPK3, MLKL, and p-MLKL. A, Quantification of Ripk1, Ripk3, and Mlkl mRNA from 12-week-old mice. Gapdh: housekeeping gene. A significant increase was detected for Ripk3 in Tg SOD1^G93A compared to Tg SOD1^WT (p = 0.0021) and NTg (p = 0.0076) at 12 weeks. B, Quantification of Ripk1, Ripk3, and Mlkl mRNA in spinal cords of 15-week-old mice. Gapdh: housekeeping gene. A significant increase was detected for Ripk1 (p = 0.0009, vs Tg SOD1^WT; p = 0.0020, vs NTg) and Ripk3 (p = 0.0115; vs Tg SOD1^WT, p = 0.0067; vs NTg) but not for Mlkl in Tg SOD1^G93A compared to Tg SOD1^WT and NTg mice at 15 weeks. C, Western blotting (RIPA) for RIPK1 in spinal cord of NTg, Tg SOD1^WT, and Tg SOD1^G93A 15-week-old mice. β-ACTIN, GAPDH: loading control. Specificity of the RIPK1 band was confirmed following downregulation of RIPK1 with specific lentiviral shRNA in mouse PMN cultures (mPMNs). D, Quantification of RIPK1 protein levels. RIPK1 protein is significantly increased in Tg SOD1^G93A samples compared to Tg SOD1^WT (p = 0.0279) and NTg (p = 0.0033) mice. Results are presented as mean ± SEM. Statistical analysis was performed via one-way ANOVA followed by Tukey’s post hoc analysis; n = 3 biological replicates per genotype. E, Western blotting (RIPA) for RIPK3 showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1^G93A). Non-specific band at 47 kDa is designated as an asterisk (*). NTg spleen: positive control tissue, Ripk3^−/− spleen and Tg SOD1^G93A;Ripk3^−/−spinal cord: negative control tissue. F, Western blotting (urea) for RIPK3 antibody showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1^G93A). Ripk3^−/− spinal cord: negative control tissue. G, Western blotting (RIPA) for MLKL showed no specific signal at the expected 55 kDa in spinal cord. NIH 3T3: positive control cell lysate. H, Western blotting (urea) for MLKL showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1^G93A). NIH 3T3: positive control cell lysate. I, Western blotting for p-MLKL (RIPA) showed no signal at the expected 55 kDa. TSZ-treated L929 cells: control cell lysate.