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Fig. 5. Ethanol-induced membrane blebbing requires RhoA-dependent, caspase-3-independent, ROCK-I activation. (A) In vitro kinase assay (measured by Histone H1 phosphorylation), shows that ethanol (100 mM) induces activation of ROCK-I in both situations: when ROCK-I was immunoprecipitated with either H-85 or C-19 antibodies. (B) Activated Rho, from lysates of astrocytes (control or exposed to ethanol), was pulled down by GST-TRBD (Rho-binding domain from Rhotekin) on glutathione beads, and analyzed by immunoblotting. (C) Lysates of astrocytes were subjected to immunoprecipitation with anti-ROCK-I (C-19) and then immunoblotted with RhoA and ROCK-I (C19) antibodies. (D) Inhibition of ROCK-I kinase activity using C3 exoenzyme in control- and ethanol-exposed astrocytes (100 mM) (see Materials and Methods). *P<0.05 versus ethanol-treated cells in a one-way ANOVA. (E-a) A representative western blotting of ROCK-I using H-85 antibody is shown, in control and ethanol-treated astrocytes. (E-b) A representative western blot analysis of PARP cleavage in astrocytes exposed to ethanol (100 mM) for 6 and 14 hours. (F) Ethanol-induced blebbing is abolished when cells are pretreated with either Y-27632 (a ROCK inhibitor) or C3 exoenzyme (a Rho inhibitor). **P<0.001 versus ethanol-treated experiments in a one-way ANOVA. (G) Preincubation of astrocytes with either Y-27632 or C3 before ethanol treatment (3, 6, 14 and 24 hours) decreases the caspase-3 activity. Percent of caspase-3 inhibition was calculated by comparing the activity in the presence and absence of the inhibitor, at each time-point analyzed. Results are mean ±s.d. of three different experiments. (H) Quantification of annexin-V positive cells (7-AAD–) of ethanol-exposed astrocytes, in the presence and absence of the ROCK inhibitor Y-27632. Results are the average of around 500 cells from three different cultures. Values are mean ±s.d. of five different experiments. *P<0.05 and **P<0.001 versus control values in one-way ANOVA.
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