Fig. 2.

Upregulation of Sur1-Trpm4 channels in astrocytes in EAE. a–e White matter of lumbar spinal cord sections from control (CTR) (a), pid-10 (b), pid-30 (c–e) WT/EAE (b, c, e), and Abcc8−/−/EAE (d) mice, immunolabeled for Sur1 and co-labeled for Trpm4; merged images are shown in (a–d and e, right panel), demonstrating extensive co-localization (yellow) of Sur1 and Trpm4; secondary antibodies were conjugated with Alexa Fluoro 488 (green) or Cy3 (red). f Lumbar spinal cord section from a pid-30 WT/EAE mouse, immunolabeled for Sur1 (left panel), and co-labeled for Trpm4 (middle panel); immunoFRET imaging is also shown (right panel), demonstrating co-assembly of Sur1 and Trpm4; secondary antibodies were conjugated with Cy3 (red) or Cy5 (purple). g Box plots (left panel) showing the percent of white matter with Trpm4 immunopositivity under control conditions (pid-0) and at pid-10 and pid-30; bar graph (right panel) showing Pearson’s correlation coefficient for Sur1 and Trpm4 co-localization; five mice/group. h Immunoblot (left panel) showing upregulation of Sur1 in WT/EAE compared to control; HSC-70 used as a loading control; co-immunoprecipitation (right panel), with immunoisolation performed using anti-Sur1 antibody, and immunoblot performed using anti-Trpm4 antibody, showing co-assembly of Sur1 and Trpm4 exclusively in EAE. The results illustrated are representative of findings in five mice/group; scale bars, 100 μm (a–d); 200 μm (e); 50 μm (f)