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. 2004 Mar 17;24(11):2837–2845. doi: 10.1523/JNEUROSCI.4789-03.2004

Figure 5.

Figure 5.

IL-1β regulates astrocytic phenotype via deactivation of Rho GTPases. a, Astrocyte cultures treated with the ROCK inhibitor Y-27632 10 μm for 24 hr, or left untreated (Ctrl), were imaged by scanning electron microscopy. Scale bar, 25 μm. Cells treated with Y-27632 developed a spherical cell body and multiple highly branched processes, but did not develop prominent microvilli (inset; scale bar, 5 μm). Data shown are representative of three experiments using astrocytes from three different brains. b and c are confocal images of astrocytes treated as in a, stained with phalloidin (red) and antibodies (green) specific for vinculin (b), or phospho-myosin light chain 2 (Ser19) (c), and imaged by confocal microscopy. Results illustrated are representative of three independent experiments using astrocytes from three different brains. Scale bars, 40 μm. d and e show immunoblots comparing levels of vinculin, phospho-focal adhesion kinase (Tyr576/577), phospho-myosin light chain 2 (Ser19) (d) and phospho-myosin phosphatase targeting subunit (Thr696) (e) in total lysates from astrocyte cultures treated with 10 ng/ml IL-1β or 10 μm Y-27632 for times shown. The experiment illustrated is representative of three experiments using astrocytes from three different brains in all cases. f, Astrocyte cultures treated with 10 ng/ml IL-1β for times shown were subjected to pulldown assay with rhotekin RBD, which binds the active form of Rho GTPase. Levels of total Rho in the same samples were also assessed in parallel (bottom blot). The experiment illustrated is representative of three independent experiments using astrocytes from three separate brains. g, Astrocytes were treated with IL-1β for times shown, or with EGF (positive control, lane marked +), and blots of total cell lysates were probed with an antibody specific for phospho-Rac1/cdc42(Ser71). Data shown is representative of three independent experiments using astrocytes from three different brains. h compares the effect of IL-1β on astrocytic morphology in cultures transfected with constitutively active Rho (G14V) versus empty vector-transfected controls. Constitutively active Rho significantly inhibited the effects of IL-1β on astrocytic morphology (p < 0.0001). Data shown is from an individual experiment using triplicate samples from the same brain for each condition and is representative of three independent experiments using astrocytes from three different brains.