Figure 5. Ethanol mediated inhibition of IL-1β secretion involves inhibition of ROS production and is dependent on protein tyrosine phosphatase activity.

ROS production measured by a DCF-DA assay of J774 cells treated with ethanol for 30 minutes (A), LPS (1μg/ml) for 5 h and ethanol and ATP (1mM) for 30 minutes (B). A western blot probing for mouse phosphotyrosine residues in the lysates of J774 cells primed with LPS (37ng/ml) for 8 h and treated with ethanol (3%), and ATP (5mM) for 1 h (C), or treated with LPS (37ng/ml) for 8 h, pre-treated with Na₃VO₄ (10–1000μM) for 1 h prior to ethanol (3%) and ATP (5mM) for 1 h (D). An IL-1β and TNF ELISA from the supernatants of J774 cells primed with LPS for 8 h and pre-treated with Na₃VO₄ for 1 h prior to the addition of ethanol and ATP for 1 h (E). A western blot probing for p-ASC (Y144) and ASC in lysates from J774 cells treated with LPS (37ng/ml) for 7 h, Na₃VO₄ (1mM) for 1 h, and ethanol (3%) for 1 h (F). A western blot probing for p-ASC (Y144) in the supernatants of J774 cells treated with LPS (37ng/ml) for 8 h, and EtOH (3%) and ATP (5mM) for 1 h (G). *<0.0001 by a Tukey’s post hoc test following a one-way ANOVA relative to the LPS+ATP treated group. EtOH = ethanol, Na₃VO₄ = sodium orthovanadate, RFU = relative fluorescence units, AUC = area under the curve. All studies were performed in triplicate.