Fig. 7

OPTN negatively regulates RIPK1 inflammatory signaling pathways. A BV2 cells with OPTN silenced were treated with Aβo for 12 h. Then, OPTN, RIPK1, p-IκBα, and IκBα in the cytoplasm and NF-κB in the cytoplasm or nucleus were detected by western blot with β-actin as an internal control. B–G ImageJ software was used to semiquantitatively analyze the optical density of western blots. H–K OPTN-silenced BV2 cells were treated with Aβo for 12 h. H OPTN mRNA expression was detected by qRT-PCR using GAPDH as an internal control. I RIPK1 RNA expression was detected by qRT-PCR using GAPDH as an internal control. J Extracellular secretion of IL-1β was assessed by ELISA. K The binding activity of NF-κB was evaluated by dual-luciferase assay. The data are presented as the means ± S.M. of independent experiment. OPTN-silenced BV2 cells compared to control BV2 cells or Aβo-treated BV2 cells compared to vehicle BV2 cells, *P < 0.05, **P < 0.01, ***P < 0.001. L–V BV2 cells with ectopic overexpression of OPTN in the absence or presence of Aβo treatment for 12 h. L Protein levels of OPTN, RIPK1, p-IκBα, and IκBα in the cytoplasm and NF-κB in the cytoplasm or nucleus were detected by western blot using β-actin as an internal control. N–R ImageJ software was used to semiquantitatively analyze the western blot results. S mRNA expression of OPTN was detected by qRT-PCR using GAPDH as an internal control. T mRNA expression of RIPK1 was detected by qRT-PCR using GAPDH as an internal control. U Extracellular secretion of IL-1β was assessed by ELISA. V The binding activity of NF-κB was evaluated using a dual-luciferase assay. The data present means ± S.M. of independent experiment. OPTN overexpressed BV2 cells compared with control BV2 cells or Aβo-treated BV2 cells compared with vehicle BV2 cells, *P < 0.05, **P < 0.01, ***P < 0.001