Figure 6.
The stimulatory effect of A2BAR activation on IL-10 production by microglia is CREB-dependent. A. BV-2 cells were treated with 10 μM NECA and 20 μg/ml PGN for 30 min and ChIP assay was performed on cell extracts using phospho-CREB-specific and control antibodies. PCR reaction was performed using the DNA purified from ChIP samples using primers specific for the IL-10 promoter. PCR products were separated on a 3% agarose gel stained with ethidium bromide and visualized under UV light. B. BV-2 cells were transduced with lentiviral vectors containing CREB-specific or non-targeting (NT) shRNA. Protein was then isolated from transduced and untransduced BV-2 cells, and CREB expression was tested by Western blotting using antibodies specific for CREB or for actin as loading control. C. BV-2 cells transduced with NT or CREB-specific shRNA were treated with 10 μM NECA and 20 μg/ml PGN and incubated for 24 hours. IL-10 concentrations were determined from the supernatants that were taken at the end of the incubation period using ELISA. Data are expressed as percent of vehicle (treated only with PGN). ***p<0.001 vs. vehicle, ###p<0.001 vs. NECA/NT shRNA-transfected group. D. BV-2 cells were treated with 0.1 μM isoproterenol or PGE2 and 20 μg/ml PGN for 24 hours and IL-10 concentrations were determined from the supernatants. ###p<0.001 vs. PGN. Results (mean ± SEM) shown are representative of three separate experiments (n=4 in each experiment).