Figure 8
From: Anisomycin prevents OGD-induced necroptosis by regulating the E3 ligase CHIP
CHIP functions as an anti-necroptotic factor in a domain-dependent manner (A) N2a Cells transfected with wild-type CHIP and different domain mutants of CHIP (K30A, T246M) were analyzed for ratio of PI positive cells and apoptosis cells by Flow cytometry after OGDR (B) PI positive cells were analyzed. Bars represent the mean ± SEM of four independent experiments. Significant differences #p < 0.05 vs. Control, *p < 0.05 vs. Vector. (C) Apoptosis cells were analyzed. Bars represent the mean ± SEM of four independent experiments. Significant differences #p < 0.05 vs. OGD, *p < 0.05 vs. Vector. (D) Immunofluorescence showing expression of RIPK3 (red) and MLKL (green) in cells transfected as above. Nuclei are stained in blue. Scale bar, 10 μm. (E,F) Representative western blot (left hand panel) of cells transfected with CHIP and CHIP mutants showing the expression of CHIP, RIPK3, MLKL and p-MLKL. The right-hand panel showed a semi-quantitative analysis using β-actin for normalization (bars represent the mean ± SEM of four independent experiments). *p < 0.05 vs. Vector, #p < 0.05 vs. CHIP. Related blots are shown in Supplementary Fig. S7.
