Fig 1: HK2 directly binds to and phosphorylates nSMase1. A) Protein expression of HK2, nSMase1 and nSMase2 in astrocytes transfected with siHK2‐1 and siHK2‐2. B,C) Quantification of HK2 B) and nSMase1 C) protein levels normalized to β‐actin and presented as fold‐change relative to the control group in (A). D) The immunofluorescence imaging of nSMase1 and HK2 in human astrocyte cell line. The fluorescence intensity curve of HK2 (TRITC) and nSMase1 (FITC) was shown to indicate the colocalization of HK2 and nSMase1 in primary human astrocytes, bar = 20 µm (up), bar = 10 µm (down). E) CO‐IP confirmed that nSMase1 interacted with HK2 under hypoxia. F) Relative nSMase1‐bound HK2 normalized to the control group. G) The structural scheme of FLHK2 and MuHK2. H) Representative immunofluorescence images of Flotillin‐1‐labeled ILVs (green) in primary human astrocyte (GFAP, red) transfected with empty vector, FLHK2 or MuHK2 vectors, bar = 10 µm. I) Statistical analysis of ILVs numbers of astrocytes in (H). J) Phos‐tag western blot of cell lysates of human astrocytes transfected with empty vector, FLHK2 or MuHK2 vectors. K,L) Quantification of phos‐nSMase1 band intensity (normalized to FL‐HK2 group) was performed under control K) and hypoxia L) conditions. (M and N) HEK293T cells transfected with pcDNA3.1‐His‐nSMase1 were lysed and subjected to immunoprecipitation using anti‐His magnetic beads, and then purified His‐nSMase1 protein was incubated with varying amounts of recombinant HK2 protein in vitro M) or with/without lonidamine N) at 37 °C for 1 h. The reaction mixtures were then subjected to Western blotting and probed with anti‐Phosphoserine/threonine/tyrosine antibody. Data were presented with Mean ± SEM. Statistical analysis in (F), (K) and (L) was performed by unpaired Student's t test. Statistical analysis in (B), (C) and (I) was performed using one‐way ANOVA followed by Tukey's multiple comparison tests. n.s., no significance. * p < 0.05; ** p < 0.01; *** p < 0.001.