Figure 3

Anisomycin treatment increased CHIP expression while reducing necroptosis in OGD-challenged N2a cells. (A) Cells managed as Fig. 2. 12 h recovery after OGD, the relative protein levels of total JNK, phospho-JNK, RIPK3, p-MLKL and CHIP were measured by western blotting (left-hand panel, representative blot). (B) Semi-quantitative analysed of relative proteins (right-hand panel, bars represent the mean ± SEM of eight independent experiments). Significant differences *p < 0.05 vs. Control, ^#p < 0.05 vs. OGD. (C) 24 h prior to an OGD challenge, N2a cells were treated with or without 100 nM anisomycin, or 10 μM SP600125. After a 12 h recovery, q-RT-PCR was used to quantitate the relative mRNA levels of CHIP. The bars represent the mean ± SEM of five independent experiments. Significant differences *p < 0.05 vs. OGD. (D) 24 h prior to an OGD challenge, N2a cells were treated with or without 100 nM anisomycin, or 10 μM SP600125. After a 12 h recovery of OGD, q-RT-PCR was used to quantitate the relative mRNA levels of RIPK3. The bars represent the mean ± SEM of five independent experiments. Significant differences *p < 0.05 vs. Control, ^#p < 0.05 vs. OGD. Related blots are shown in Supplementary Fig. S2.