Lv scramble

Lentiviruses containing the ABIN1 shRNA (LV-shABIN1) for ABIN1 knockdown and control shRNA (LV-Scramble) were supplied by Genechem (Shanghai, China). Two weeks prior to the establishment of the MCAO/R model, the right cortex of rat was injected with the lentiviruses. The injection sites were as follows: site 1, A-P 1.0 mm; M-L −2.0 mm; D-V −1.2 mm, and site 2, A-P −3.0 mm; M-L −1.5 mm; D-V −1.2 mm [34 (link)]. A total of 2.5 μl of LV-shABIN1 or LV-Scramble were injected into each site.

The human neuroblastoma cell line (SH-SY5Y cells) were purchased from the American Type Culture Collection. The cells were cultured in RPMI 1640 medium (Hyclone, Thermo Fisher Scientific) containing 10% fetal bovine serum (Hyclone) in a humidified atmosphere at 37°C with 5% CO₂. Cells were incubated in neurobasal medium, supplemented with 2% B27 supplement (17504-044, Gibco) and 0.5 mM L-glutamine (35050-061, Gibco). 3 days of plating, cells were added 10 M retinoic acid (0695, Sigma) to the medium, and induced to differentiate into a homogeneous population of the cells with neuronal morphological structure (Kume et al., 2008 (link)). After 7–8 days, cells were used in the experiments.
The lentivirus for knock down NLRP3 and the control lentivirus (LV-scramble) was synthesized by Genechem (Shanghai, China). A plasmid expressing shRNA that targeting human NLRP3 was constructed using the GV248 vector. The shRNA target sequence was designed follows: 5′-GTGCGTTAGAAACACTTCA-3′. The shRNA (100 nmol/L) were transfected into the cells using Lipofectamine 2000 reagent (11668, Invitrogen) according to the manufacturer’s instructions. After 6 h of transfection, the medium was replaced by RPMI 1640 medium for 24 h and then treated with or without OGD deprivation.