Research Paper Volume 11, Issue 18 pp 7723—7745

Neural stem cell small extracellular vesicle-based delivery of 14-3-3t reduces apoptosis and neuroinflammation following traumatic spinal cord injury by enhancing autophagy by targeting Beclin-1

Figure 8. 14-3-3t enhanced autophagy by interacting with Beclin-1. (A) Western blot assay for Beclin-1 in neuronal cells. (B) Western blot analysis of Beclin-1 in the Ad-GFP-sEVs and Ad-14-3-3t-sEVs groups in neuronal cells. (C) The expression of Beclin-1 proteins in neurons of the Ad-GFP-sEVs and Ad-14-3-3t-sEVs groups was detected by immunofluorescence. Scale bar = 50um. (D) Following the overexpression of flag-14-3-3t (from adenovirus) in neuronal cells, anti-Flag antibody was added. Co-immunoprecipitation results showed that 14-3-3t interacts with Beclin-1 in neuronal cells. (E) Indirect in situ PLAs were used to visualize protein interactions using red fluorophore-labeled oligonucleotides. Those assays showed that endogenous Beclin-1 interacted and co-localized with 14-3-3t in neurons following Glu treatment. Scale bar = 20um. Glu, Glutamate; PLA, Proximity ligation assay.