Figure 1.

An NADPH oxidase is expressed in primary astrocytes. A, Western blots showing the expression of the subunits of the NADPH oxidase. i shows a Western blot of whole-cell extracts showing the expression of gp91phox and p67phox in astrocytes (a) using neutrophils (n) as a reference. PNS material from rat neutrophils and astrocytes was obtained and subjected to Western blot analysis as described in Materials and Methods. Six micrograms of neutrophil and 30 μg of astrocyte protein was applied and probed with antibodies to human gp91phox and p67phox as shown, because there is a high degree of homology between the proteins from human and rat. Arrowheads indicate the positions of the proteins. The figure is representative of three experiments. ii, Cytosol proteins probed for the cytosolic components of the NADPH oxidase, showing the expression of p67phox, p47phox, p40phox, and Rac as indicated. iii shows expression of the flavocytochrome subunits in the membrane fraction, namely gp91phox and p22phox as indicated. For a control and to ensure specificity, a control blot was made using antigp91phox antiserum, which was depleted for anti-gp91phox antibodies by adsorbing them out; this is not shown because the blot was completely blank. B, Immunofluorescence imaging using antibodies to gp91phox and p67phox counterstained with Cy5-labeled rabbit anti-human antibodies reveal the expression of both proteins in astrocyte cultures. Cells were counterstained with antibodies to GFAP (green) as a glial-specific marker and with DAPI to stain nuclei (blue), and images were acquired on a Zeiss (Welwyn Garden City, UK) 510 CLSM. These images are representative of five separate preparations, and staining was invariably seen clearly. The bottom panel shows a slightly higher gain image to show the distribution of the gp91phox. Appropriate controls showed no significant background signal under these conditions. Scale bars, 20 μm.