Fig. 8. Altered response to external stimuli in double null mutant cells. (A) Primary astrocytes were stimulated with EGF (25 ng/ml; upper panel) or LPS (1 µg/ml; lower panel) and analyzed for p38 MAPK phosphorylation using anti-phospho-p38 MAPK antibodies. The filters were subsequently stripped and probed with anti-p38 MAPK antibodies. The histograms to the right represent the relative quantitation of the scanned images. In the histograms, the wild-type (control) phospho-p38/p38 ratio without stimuli was arbitrarily set at 1 (upper histogram, average of two independent experiments; lower histogram, n=4, error bars indicate s.d.). Blue bars, wild-type samples; red bars, double null mutant samples. (B,C) Comparison of serum-starved and EGF-stimulated wild-type control and double null mutant primary astrocytes. Primary astrocytes from wild-type controls and double null mutants were serum-starved for 24 hours (left panels) or stimulated with EGF (25 ng/ml) for 24 hours (right panels). Astrocytes were identified by labeling for GFAP (red-Cy3), double stained for actin (green-FITC) and analyzed using confocal microscopy. Serum-starved null mutant astrocytes were more spread than controls. When stimulated with EGF, wild-type controls showed no morphological changes, whereas some Abr^-/-;Bcr^-/- astrocytes (arrows in C) responded with the projection of GFAP-positive protrusions. Scale bar: 20 µm.