Fig 1: TREK-2 protein is up-regulated in vivo in astrocytes after tMCAO.(A) Expression changes of TREK-2 channel protein in brain after tMCAO. The results of 4 separate tMCAO experiments are shown. The asterisk indicates a significant difference from contralateral side (control) ± SEM (t-test; p<0.05). (B) TTC staining delineates a clearly detectable lesion at the infarct core (left side) at 24 hours after 60 minutes of tMCAO. (C) Immunostaining for TREK-2 (green labeling) and GFAP (red labeling) in hippocampus after tMCAO. Representative images show a qualitative increase of TREK-2 levels in hippocampus (C) on the ipsilateral (lesion) side of the brain. White arrows point to astrocytic processes and endfeet. Insets show higher magnification of the merged image to highlight colocalization between GFAP and TREK-2 channels in astrocytes.
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).
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