Fig. 4

RGS5 is necessary for astrocytic TNFRs-promoted inflammatory factor expression with TNF-α/LPS challenge. A Double immunofluorescence staining for GFAP and TNFR1 or TNFR2 on the substantia nigra (SN). Arrows indicate the double-labeled cells. Scale bars, 5 μm. B ELISA analysis showing increased secretion of TNF-α in primary cultured astrocytes overexpressing TNF receptors mediated by lentivirus following exposure to TNF-α. The cells were incubated with TNF-α (100 ng/ml) for 1 h, and then washed 3 times with DMEM. Then, the culture medium was replaced with fresh DMEM containing 10% FBS and collected for ELISA 4 h later (n = 6). C, D Representative Western blots (C) and quantifications (D) showing endogenous TNF-α and pro-IL-1β protein expression in primary cultured astrocytes overexpressing GFP, TNFR1 or TNFR2 mediated by lentivirus transfection following exposure to TNF-α or vehicle for 5 h. The cells were washed three times with PBS before lysed. TNF-α 100 ng/ml, 5 h, n = 7. E–H Representative Western blots showing the reduction of TNF-α and pro-IL-1β protein levels in Rgs5-null (RGS5^hGFAP) astrocytes. Primary astrocytes were transfected with Lentivirus (LV-GFP, LV-TNFR1 or LV-TNFR2) followed by challenge with either TNF-α (100 ng/ml) (E, F) or LPS (500 ng/ml) (G, H). n = 3–5. Data are expressed as mean ± SEM, two tailed t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001