Fig. 1

MLKL and Cx43 were upregulated in the ipsilateral VPN after dMCAO. (A, B) Representative microphotographs of the immunofluorescence assay of MLKL (green), NeuN/CD68/GFAP (red), and DAPI (blue) in VPN of Sham-operated group and 2w after dMCAO group, respectively (scale: 50 μm). (C, D) Representative microphotographs of the immunofluorescence assay of Cx43 and NeuN/MLKL in VPN of Sham-operated group and 2w after dMCAO group, respectively (scale: 50 μm). (E) Co-localization analysis of MLKL and NeuN, CD68 or GFAP. The histogram presents the quantitative analyses of co-staining of MLKL and NeuN, CD68, or GFAP (n = 6 in each group). (F) Representative immunoblots of MLKL and Cx43 expression, respectively, in VPN after dMCAO. The histogram presents the quantitative analyses of MLKL and Cx43 protein levels (n = 3 in each group). (G, H) Representative immunoblots of MLKL and pMLKL multimers expression, respectively, in VPN after dMCAO. (I, J) Co-immunoprecipitation analysis of MLKL and Cx43. Each bar represents mean ± standard deviation. ^*P < 0.05 vs. Sham-operated group and ^#P < 0.05 vs. contralateral to the lesion after dMCAO. dMCAO, distal middle cerebral artery occlusion model; MLKL, mixed lineage kinase domain-like protein; NeuN, neural nuclear antigen; GFAP, glial fibrillary acidic protein; CD68, cluster of differentiation 68; DAPI, 4′, 6-diamidine-2-phenylindole. Cx43, connexin 43; GAPDH, glyceraldehyde 3-phosphate dehydrogenase