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Fig. 6. Ethanol-induced MLC phosphorylation correlates with ROCK-I kinase activity. Cultured astrocytes were treated with 100 mM alcohol for 3, 6, 14 and 24 hours. Lysates of control and ethanol-treated astrocytes were subjected to immunoprecipitation with the anti-ROCK-I antibody C-19. These immunoprecipitates were used for A and B. (A) ROCK-I kinase activity measured by phosphorylated Histone H1. A representative autoradiograph is shown. (B) Western blot analysis of ROCK-I, as a control of protein content. (C) A representative western blotting of phosphorylated MLC from lysates of astrocytes (control and ethanol-treated) that were immunoprecipitated with anti-MLC-pp antibody. (D) Inhibition of the ethanol-induced MLC phosphorylation when astrocytes are pretreated with either Y27632 or C3. Bars represent the mean ±s.d. of the densitometric quantification obtained from three different immunoblots. Percent of MLCpp inhibition was calculated by comparing the densitometric values in the presence and absence of the inhibitor, at each time-point analyzed.
