Fig. 2: Evidence for activation of RIPK1, RIPK3, and MLKL in brain tissue and immunopanned cells after CCI.
Mice were subjected to sham or CCI and brain tissue or cell populations isolated by immunopanning were subjected to immunoprecipitation and western blot analyses. A Representative western blot and B–D densitometry analyses of the 1% triton X100-soluble and 8 M urea-soluble brain tissue fractions from sham and CCI mice. RIPK1, RIPK3, and MLKL protein levels were increased by 24 h after CCI (*p < 0.05, **p < 0.01, n = 3/group). E Representative western blots and F densitometry analyses of phospho-RIPK3 (p-RIPK3) and p-MLKL expression in immunopanned CD31 + endothelial cells, CD11b + immune cells, and neurons isolated from sham and injured mouse brain at 3 h after CCI. p-RIPK3 was increased in CD11b + cells and neurons, p-MLKL was increased in neurons after CCI (n = 3–4/group, *p < 0.05, **p < 0.01). G Representative western blots and H densitometry analyses of phospho-RIPK3 (p-RIPK3) and p-MLKL expression in immunopanned CD31 + endothelial cells, CD11b + immune cells and neurons isolated from sham and injured mouse brain at 24 h after CCI. p-RIPK3 was increased in CD31 + cells, CD11b + cells and neurons, p-MLKL was increased in neurons after CCI (n = 3–4/group, *p < 0.05). I Representative immunoprecipitation (IP) with RIPK3 or MLKL pull down and western blot analyses for RIPK1 showing RIPK1-RIPK3-MLKL interaction early after CCI. Note lack of reactivity in RIPK3−/− and with isotype control IgG. Data are representative of three independent experiments. J Representative western blots and K densitometric analyses of three-dimensional cultures of human brain endothelial cells subjected to sham or CCI showing increased expression of pRIPK3 and total RIPK3 at 24 h (n = 3–6/group, *p < 0.05, **p < 0.01).
