Fig. 6: Lysosomal degradation contributes to regulation of necroptosis in vivo after SCI.

a Expression of RIPK1, RIPK3, and MLKL in the cytosol and at the lysosomes in mouse spinal cord. Sham and SCI mouse spinal cord tissue was fractionated to isolate cytosolic and lysosome-enriched fractions. Lysosomal membrane protein LAMP1 was used to identify lysosomal fraction and as a loading control. Full unedited western blots are presented in Supplemental Figure S3b. b Quantification of RIPK1 data from a and S3b. c Quantification of RIPK3 data from a and S3b. d Quantification of MLKL data from a and S3b. n = 6. e Western blot data demonstrating that induction of autophagy and lysosomal function with Rapamycin decreased accumulation of RIPK1 and attenuated cell death in the mouse spinal cord tissue surrounding injury site at 24 h after SCI. Full unedited western blots are presented in Supplemental Figure S8a–c. f Quantification of phospho-S6 from e demonstrating inhibition of mTOR activity by Rapamycin after SCI. g Quantification of cleaved (145–150 kDa) α-fodrin from e. h Quantification of p62/SQSTM1 levels from e. i Quantification of RIPK1 levels from e. j Quantification of MLKL levels from e. n = 6. All data are presented as mean±SE. *p < 0.05, **p < 0.01, ***p < 0.001