Adeasy adenoviral vector system kit

The above plasmids with genes of interest were used for adenovirus packaging with the AdEasy Adenoviral Vector System Kit (Agilent Technologies, Santa Clare, CA, USA) according to the manufacturer's protocol. The concentrated adenoviruses were directly used to infect host cells. The host cells were infected for 5 h in growth media.

LAPTM5-overexpressing adenovirus (AdLAPTM5) was purchased from Hanheng Biology (Shanghai, China). To construct the LAPTM5 knockdown adenovirus, hairpin-forming oligonucleotides (Primers in Table 4) were synthesized, annealed, and cloned into pENTR-U6-CMV-GFP shuttle vector. Adenoviruses were generated using an AdEasy Adenoviral Vector System Kit (Cat# 240009; Agilent Technologies). The above plasmids were recombined with the pAdEasy backbone vector and then transfected into HEK293A cells using TurboFect transfection reagent (Cat# R0531; ThermoFisher Scientific). Recombinant adenoviruses were plaque-purified using cesium chloride density gradient centrifugation and verified by restriction digestion.
For the in vitro experiments, cultured neurons were transfected with adenoviruses at a multiplicity of infection (MOI) of 100 for 48 h before OGD/R treatment.