Fig. 1

a Western blots showing that OGD/R-enhanced autophagy in WT cells was abolished in NKAα1 KO cells. Both WT and NKAα1 KO cells were subjected to OGD (3 h)/R (2 h) in the presence or absence of BafA1 (100 nM). n = 3. b qPCR analysis showing that the mRNA levels of autophagy-related proteins (AMPKα1, AMPKα2, ULK1, Atg13, Beclin1, Atg12, LC3, and p62) were significantly reduced in NKAα1 KO cells compared with WT N2a cells. n = 4. Atg13 Autophagy-related protein 13, Atg12 Autophagy-related protein 12. c Cell viability assay showing that NKAα1 loss exacerbated OGD/R-induced damage. n = 6. d Western blot analysis showing that DR-Ab treatment reversed the loss of membrane NKAα1 caused by OGD/R, while the total level of NKAα1 remained unaffected in response to OGD/R model. n = 4, DR DR-Ab, Veh vehicle, m-NKAα1 membrane NKAα1, t-NKAα1 total NKAα1. e Cell viability test showing the protective effect of DR-Ab. n = 4. Con control, Veh vehicle, DR DR-Ab. f Confocal microscopy images showing that DR-Ab significantly increased LC3 dots in N2a cells transfected with EGFP-LC3 plasmid under both normoxic and hypoxic conditions. BafA1 (100 nM) was added to each group to magnify and visualize autophagy flux. Magnification: 300×. LC3 dots were counted with ImageJ software. Data were from three independent experiments. A total of 55–110 cells were counted in each group. Scale bar: 5 μm. g Representative TTC-stained brain sections and quantitative data showing that the blockade of autophagy with 3MA abolished the protective effect of DR-Ab on the infarction volume caused by tMCAO. n = 6–7/group. 3MA 3-methyladenine, 100 nM/mouse, 2 h before ischemia, icv; DR: DR-Ab, 200 μg/mouse, 1 h before ischemia, iv; DR + 3MA: (3MA, 100 nM/mouse, 2 h before ischemia, icv) + (DR-Ab, 200 μg/mouse, 1 h before ischemia, iv). h Western blot analysis showing that NKAα1 KO reduced total and phosphorylated AMPKα levels under both normoxic and hypoxic conditions. n = 3. i Western blot analysis showing that DR-Ab significantly increased the levels of AMPKα phosphorylated at Thr172 under both normoxic and hypoxic conditions. n = 3–4. p-AMPKα phosphorylated AMPKα, t-AMPKα total AMPKα. j Representative western blots showing that OGD/R induced the dissociation of NKAα1 and AMPKα, which was further enhanced by DR-Ab treatment. Cells were immunoprecipitated with anti-AMPKα antibody, followed by NKAα1 antibody detection. n = 3. k In vitro GST pull down assay to define the specific intracellular domains of NKAα1 responsible for its direct interaction with AMPK. The purity and amount of purified GST-fused protein were shown in the lower panel. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001 as indicated. Bars represent the mean ± s.e.m. Unpaired two-tailed t-test (b) or one-way ANOVA with Bonferroni’s multiple comparison test (a, c, d, e, f, g, h, i, and j)