The NP1 induction is associated with OGD-induced cortical neuronal death. A) Total cellular RNA was extracted and NP1 mRNA expression levels were analyzed by RT-qPCR. Data show relative quantification of Nptx1 expression at different time periods of OGD exposure. Fold induction is the ratio of NP1 to internal control HPRT, which remained stable throughout the OGD period (mean ± SEM, n = 6; *p < 0.05, **p < 0.01). B) Total cellular protein was analyzed by SDS-PAGE and immunoblotted for NP1 protein using NP1-specific antibody that detected NP1-immunoreactive single band of molecular mass 47 kDa. The β-actin serves as loading control. Quantitative densitometry values normalized to β-actin (NP1/β-actin ratio, n = 6) are also shown. Representative bands are shown. C & D) OGD exposure of WT cortical neurons caused cell death, while NP1-KO neurons were protected against OGD. Quantification of cell death as indicated by LDH release showed OGD time dependent increase of LDH release at 2, 4, 6, and 8 h of OGD exposures of WT cortical neurons. LDH release remained at the control level or non-significant increase in OGD-exposed NP1-KO neurons. Data are expressed as % LDH release normalized to normoxia control (mean ± SEM, n = 8; *p < 0.05, ***p < 0.001). We found ~ 50% cytotoxicity occurred at 6 h of OGD.