Fig. 2

Penfluridol inhibits TNFα-induced NF-κB activation in vitro. BMDMs were starved with 2% FBS overnight, followed by adding DMSO or penfluridol (1 μM) for 2 h and then stimulated with TNFα (10 ng/mL) for 0, 15, 30, or 60 min. A Western blotting to test the effect of penfluridol on the phosphorylation of MAPKs induced by TNFα in BMDM isolated from WT C57BL/6 mice. B Western blot to determine the p65 translocation from cytoplasm to nucleus in BMDM. C After starving BMDMs with 2% FBS overnight, the cells were treated with DMSO or penfluridol (1 μM) for 2 h, followed by stimulation with TNFα (10 ng/mL) for 4 h, then nuclear extracts were used to test the binding activity of p65 to DNA in BMDM, detected by ELISA. D–I After adding DMSO or penfluridol (1 μM) for 2 h, BMDMs were stimulated with TNFα (10 ng/mL) for 24 h, then supernatants were collected to detect cytokine secretion levels by ELISA and cells were collected to test mRNA expression levels of cytokines by real-time quantitative PCR (qRT-PCR). D mRNA expression level of IL-1β. E mRNA expression level of IL-6. F mRNA expression level of IL-17. G mRNA expression level of NOS2. H Secretion level of IL-1β. I Secretion level of IL-6. Experiments were performed for 3 biological replications, and one-way analysis of variance and Bonferroni post hoc test were used to test statistical significance of the differences among groups (*p < 0.05, **p < 0.01, ***p < 0.001). PF: penfluridol