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. 2021 Jun 25;30(22):2082–2099. doi: 10.1093/hmg/ddab159

Figure 2 .

Figure 2

Core elements of the ripoptossome/necroptosome are upregulated in mouse models of Krabbe and SD. Spinal cord was examined as a paradigm of disease. (A) RT-qPCR of total RNA at three stages of disease in twi-2J with markers Tnf, Tnfrsf1, Ripk1, Ripk3, Mlkl, Casp8, cFlipL and Casp3; (B) Immunoblots of Ripk1, cathepsin D and caspase-8 in twi-2J and SD mice at the HEP. 293T cells transfected with a plasmid expressing murine caspase-8 was used as a positive control for the antibody against caspase-8. One hundred microgram protein homogenate was loaded per lane. Blots were stripped of first antibody and re-probed; (C) Densitometry of Ripk1 relative to α-tubulin in B. Note, of the cleaved Ripk1 species only the lower one was used for densitometry as the upper form is often difficult to detect in wt animals. (D) RT-qPCR of total RNA from SD at HEP with markers: Tnfrsf1, Ripk1, Ripk3, Mlkl, Casp8 and Casp3. Tumour necrosis factor (Tnf), Tumour necrosis factor receptor 1 (Tnfrsf1), Receptor-interacting serine-threonine kinase 1(Ripk1), Receptor-interacting serine-threonine kinase 3 (Ripk3), Mixed lineage kinase domain-like (Mlkl), Caspase-8 (Casp8), Caspase-3 (Casp3). mutant (mut), wild type (wt). Student’s t-test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.