Figure 7.

Akt phosphorylation plays a critical role in STS-mediated inhibition of OGD/R-induced JNK1/2 and NF-κB activation, subsequent TNF-α expression, positive NF-κB/TNF-α feedback loop, and TNF-α amplification. (A and B) Cells were exposed to OGD/R or TNF-α with or without STS. Total protein (30 μg) was analysed by Western blot with antibodies specific for p-Akt or Akt, with β-actin as the loading control. Results are representative of three independent experiments. (C–E) Cells were incubated with SP600125 (10 μM), SB203580 (10 μM), LY294002 (20 μM) or wortmannin (500 nM) for 30 min, followed by 6 h OGD and 4 h recovery with or without STS. Whole-cell lysates were examined using Western blot with the indicated antibodies. Results are representative of three independent experiments. (F) Cells were incubated with the LY294002 (20 μM) or wortmannin (500 nM) for 30 min, and subjected to 6 h OGD and 18 h recovery with or without STS. Cell lysates and cytoplasmic and nuclear extracts were prepared, and the levels of cytoplasmic, nuclear and total p65 protein determined using Western blot. Results are representative of three independent experiments. (G) The specific DNA-binding activity for nuclear NF-κB was analysed with EMSA. The graph depicts results from one of three independent experiments. (H) TNF-α and β-actin mRNA expression levels were determined using RT-PCR. Representative results of five independent experiments are shown. (I) TNF-α release was measured with elisa. Results are expressed as mean values ± SD of three independent experiments performed in triplicate. ^###P < 0.001, significantly different from control, ***P < 0.001, significantly different from OGD/R alone, ^§§§P < 0.001, significantly different from OGD/R + STS. (J and K) Translocation of NF-κB and TNF-α expression were examined to determine the effects of STS on the positive feedback loop of NF-κB/TNF-α. Results are representative of three independent experiments.