Fig 2: TREK-2 protein degradation pathway(s) are not altered after ischemia.Cortical astrocyte in cultures exposed to (A) control or (B) hypoxia/hypoglycemic conditions for 24 hours were treated with either 10 µM chloroquine, 50 µM calpeptin or 10 µM MG132 for 8 hours. Astrocytes were initially exposed to hypoxia/hypoglycemic conditions for 16 hours. The cells were then treated with the degradative pathway inhibitors or without treatment and returned to hypoxia/hypoglycemic conditions for an additional 8 hours. TREK-2 expression in astrocytes was determined by Western blot. The results of 3 separate experiments using different astrocyte cultures are shown. Data are expressed relative to control.

Fig 3: TREK-2 expression is increased in the astrocytic membrane and cytoplasm after ischemia.Cortical astrocytes in culture were exposed to hypoxic/hypoglycemic conditions for 24 hours and then processed using a cell surface biotinylation assay. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM) of TREK-2 protein in membrane and cytoplasmic fractions obtained from control astrocytes and astrocytes subjected to ischemia (representative Western blots shown below the graph). TREK-2 was detected as a band of around 60kDa which is consistent with the predicted molecular weight of 59.6kDa. The results of 4 separate experiments using different astrocyte cultures are shown. The asterisks indicate a significant difference from control (t-test; p<0.05). Data are expressed relative to control.

Fig 4: TREK-2 up-regulation in response to ischemia is due to De novo protein synthesis.Cortical astrocytes were treated with different protein synthesis inhibitors (1µg/mL cycloheximide, 300nM emetine, 5µg/mL puromycin) or without treatment (vehicle) for 24 hours. The cells were then exposed to control or hypoxia/hypoglycemic conditions for 24 hours still in the presence of the inhibitors. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. The graph summarizes the effect of three different protein synthesis inhibitors on TREK-2 up-regulation during ischemia. The data are expressed as relative chemiluminescence intensity ± SEM. The results of 6 separate experiments using different astrocyte cultures are shown. The asterisk indicates significant difference from the corresponding control (ANOVA followed by Tukey’s test; p<0.05).