Figure 4

Anisomycin upregulates CHIP and functions as an anti-necroptotic factor in OGD-challenged primary cultured hippocampal neurons. Primary cultured hippocampal neurons were treated with or without 50 nM anisomycin, or 10 μM SP600125 at the 7th day of culture in vitro. (A) After 4 h OGD insulted and recovery for 12 h, lysates from the above cells were harvested. The indicated proteins were detected by Western blot. The protein levels of total JNK, phospho-JNK, RIPK3, p-MLKL and CHIP were measured by western blotting (left-hand panel, representative blot) and semi-quantitative analysed (right-hand panel, bars represent the mean ± SEM of eight independent experiments). (B) Semi-quantitative analysed (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, cells were treated with or without 50 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, cells were treated with or without 50 nM anisomycin, or 10 μM SP600125. After a 12 h recovery, 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. S3.