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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: 6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation

Fig. 3

6-MP decreases LPS-induced transactivation of NF-κB and TNF-α promoter. BV-2 cells were preincubated with vehicle or 6-MP (50 μM) for 16 h before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. a IκB-α degradation is not suppressed by 6-MP. Western analysis was used to determine total and phosphorylated IκB-α proteins in whole cell extracts. b Translocation of p65 from cytosol to the nucleus was determined by immunoblotting. β-Actin and histone deacetylase 1 (HDAC1) immunoblotting were performed to monitor loading for cytosol and nuclear proteins, respectively. c Putative 4-κB sites within the mouse TNF-α promoter (κB1 to 4) and a consensus κB sequence are schematically depicted. The underlines indicate the binding sequences for NF-κB. d Cells were pretreated with vehicle or 6-MP (50 μM) for 16 h before stimulation with 100 ng/ml LPS for 60 min. Nuclear proteins were isolated. ELISA-based measurement of p65 DNA binding was analyzed as described in the “Methods” section. e, f Cells were transfected with a 4XNF-κB (e) or TNF-α promoter (f) luciferase construct; 24 h post-transfection, cells were preincubated with vehicle or various concentrations of 6-MP for 16 h before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for four independent experiments. *p < 0.05; **p < 0.01 compared with LPS alone

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