Fig. 4

Hypoxic preconditioning blocks the tGCI-induced plasma membrane translocation of p-MLKL and calcium influx in CA1. A Representative photomicrographs with fluorescent staining of p-MLKL (red), NeuN (green), and DAPI (blue). B, C Representative immunoblots and quantitative data show p-MLKL in plasma membrane and cytosolic proteins, respectively. D Immunoprecipitation and western blot assays showing the effects of hypoxic preconditioning on the formation of RIP3-p-MLKL (D, a) and the expression of RIP3 and p-MLKL (D, b) after tGCI. Quantification of the relative precipitated proteins is expressed as a ratio of precipitates to input. E Immunoprecipitation and western blot assays showing the effects of MLKL siRNA on the formation of RIP3-p-MLKL (E, a) and the expression of RIP3 and p-MLKL (E, b) after tGCI. F Effects of pretreatment with MLKL siRNA on the p-MLKL expression in plasma membrane protein in CA1 of tGCI rats using immunoblot analysis. G Representative intracellular calcium concentration in CA1 after tGCI with or without hypoxia. Representative images show Sham group (a), 48 h after reperfusion of tGCI group (b) and hypoxic preconditioning group (c), respectively. Quantitative analysis of fluorescence intensity of Fluo-4-AM in CA1. H Representative intracellular calcium concentration in CA1 after tGCI with or without MLKL siRNA administration. Sham group (a), tGCI+MLKL-siRNA group, infusion with MLKL-siRNA at 24 h before tGCI (b), HPC+MLKL-siRNA group, infusion with MLKL-siRNA at 24 h before tGCI with hypoxia (c). Data are expressed as percentage of value of Sham animals. Each bar represents the mean±S.D. ^*p<0.05 vs. Sham animals, ^#p<0.05 vs. tGCI groups at the same time point, and ^&p<0.05 vs. tGCI or hypoxic preconditioning group with injection of MLKL siRNA. IP immunoprecipitation, IB immunoblotting, HPC hypoxic preconditioning