Fig. 6.

Interrupting RGS5–TNFR interaction suppresses astrocytic TNF-α production. A Representative Western blots showing TNF-α expression in primary cultured astrocytes transiently transfected with GFP, RGS5, N-RGS5 or C-RGS5. TNF-α (100 ng/ml) was added 48 h after transfection and incubated for 5 h. B Quantitative data shown in A. n = 5–8. C Representative Western blots showing a marked reduction of RGS5-mediated TNF-α expression by N-RGS5. Primary astrocytes were transfected with LV-GFP, LV-RGS5 or LV-N-RGS5. TNF-α (100 ng/ml) was added 72 h after transfection and incubated for 5 h. D Quantitative data shown in C. n = 5. E The structural formula of feshurin and butein. F NanoBit assay showing that treatment with feshurin or butein interrupts RGS5–TNFR2 interaction in a dose-dependent manner. Cells were incubated with feshurin or butein for 6 h at indicated concentrations. # indicates comparisons between vehicle (DMSO) and butein-treated groups; * indicates comparisons between vehicle (DMSO) and feshurin groups. (n = 4). G NanoBit assay showing that treatment with feshurin or butein interrupts RGS5–TNFR1 interaction at indicated concentrations. (n = 4–5). H, I Reduced pro-IL-1β and TNF-α expression in astrocytes pre-treated with feshurin or butein (50 µM) for 2 h and then challenged with TNF-α (20 ng/ml, 4 h) (n = 5). Data are expressed as mean ± SEM. Two-way ANOVA followed with Bonferroni’s multiple comparisons test or two-tailed t- test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001