Figure 5

Effect of LPS on the density of A _2A R in N9 microglial cells. (A) Representative blot of the LPS (100 ng/mL for 6 hours)-mediated increase of A_2AR density; the blot compares A_2AR immunoreactivity from cells exposed (+) or not (−) to LPS in four independent experiments. (B) Cells were exposed to LPS (100 ng/mL for 6 hours) and then lysed and homogenized for Western blot analysis. Quantitative analysis of A_2AR immunoreactivity in the presence of LPS was compared with non-treated cells (taken as 100%). Results are expressed as mean ± SEM of n (as indicated in each bar) independent experiments (* P < 0.05, compared with non-treated cells, using Student’s t test). (C) Immunocytochemistry confirmed the LPS-induced increase in A_2AR immunoreactivity (representative images of independent experiments). Double-labeling for A_2AR (green) and for the structural protein, phalloidin (far red), was performed in all experiments; phalloidin-labeling is not shown to allow a clear visualization of A_2AR immunoreactivity (the nuclei were labeled with DAPI, in blue). (D) Magnification of cellular elements 1 (microglia in basal conditions) and 2 (activated cell) in (C), showing in detail the increase in A_2AR density in activated cells (phalloidin-labeling is shown to allow the visualization of LPS-induced morphological changes of microglial cells). A_2AR, A_2A receptor; LPS, lipopolysaccharide; SEM, standard error of the mean.